Methods of treating myelodysplastic syndrome with anti-TNF antibodies

ABSTRACT

Anti-TNF antibodies, fragments and regions thereof which are specific for human tumor necrosis factor-α (TNFα) and are useful in vivo diagnosis and therapy of a number of TNFα-mediated pathologies and conditions, as well as polynucleotides coding for murine and chimeric antibodies, methods of producing the antibody, methods of use of the anti-TNF antibody, or fragment, region or derivative thereof, in immunoassays and immunotherapeutic approaches are provided.

RELATED APPLICATIONS

[0001] This application is a divisional of U.S. application Ser. No.09/927,703, filed Aug. 10, 2001, which is a continuation of U.S.application Ser. No. 09/756,398, filed Jan. 8, 2001, which is adivisional of U.S. application Ser. No. 09/133,119, filed Aug. 12, 1998,now U.S. Pat. No. 6,277,969, which is a divisional of U.S. applicationSer. No. 08/570,674, filed Dec. 11, 1995, which is acontinuation-in-part of U.S. application Ser. No. 08/324,799, filed Oct.18, 1994, now U.S. Pat. No. 5,698,195, issued Dec. 16, 1997, which is acontinuation-in-part of U.S. application Ser. Nos. 08/192,102, now U.S.Pat. No. 5,656,272, issued Aug. 12, 1997, 08/192,861, now U.S. Pat. No.5,919,452, issued Jul. 6, 1999, and 08/192,093, now U.S. Pat. No.6,284,471, all filed on Feb. 4, 1994 which are continuations-in-part ofU.S. application Ser. No. 08/010,406, filed Jan. 29, 1993, nowabandoned, and U.S. application Ser. No. 08/013,413, filed Feb. 2, 1993,now abandoned, which is a continuation-in-part of U.S. application Ser.No. 07/943,852, filed Sep. 11, 1992, now abandoned, which is acontinuation-in-part of U.S. application Ser. No. 07/853,606, filed Mar.18, 1992, now abandoned, which is a continuation-in-part of U.S.application Ser. No. 07/670,827, filed Mar. 18, 1991, now abandoned.Each of the above applications are entirely incorporated herein byreference.

BACKGROUND

[0002] 1. Field of the Invention

[0003] The present invention in the field of immunology and medicinerelates to anti-tumor necrosis factor (TNF) antibodies, anti-TNFpeptides and nucleic acids encoding therefor, and to pharmaceutical anddiagnostic compositions and production, diagnostic and therapeuticmethods thereof, and to methods for treating human TNF-mediatedpathologies.

[0004] 2. Description of the Background Art

Tumor Necrosis Factor

[0005] Monocytes and macrophages secrete cytokines known as tumornecrosis factor-α (TNFα) and tumor necrosis factory (TNFβ) in responseto endotoxin or other stimuli. TNFα is a soluble homotrimer of 17 kDprotein subunits (Smith, et al., J. Biol. Chem. 262:6951- 6954 (1987)).A membrane-bound 26 kD precursor form of TNF also exists (Kriegler, etal., Cell 53:45-53 (1988)). For reviews of TNF, see Beutler, et al.,Nature 320:584 (1986), Old, Science 230:630 (1986), and Le, et al., Lab.Invest. 56:234.

[0006] Cells other than monocytes or macrophages also make TNFα. Forexample, human non-monocytic tumor cell lines produce TNF (Rubin, etal., J. Exp. Med. 164:1350 (1986); Spriggs, et al., Proc. Natl. Acad.Sci. USA 84:6563 (1987)). CD4+ and CD8⁺ peripheral blood T lymphocytesand some cultured T and B cell lines (Cuturi, et al., J. Exp. Med.165:1581 (1987); Sung, et al., J. Exp. Med. 168:1539 (1988)) alsoproduce TNFα.

[0007] TNF causes pro-inflammatory actions which result in tissueinjury, such as inducing procoagulant activity on vascular endothelialcells (Pober, et al., J. Immunol. 136:1680 (1986)), increasing theadherence of neutrophils and lymphocytes (Pober, et al.,J. Immunol.138:3319 (1987)), and stimulating the release of platelet activatingfactor from macrophages, neutrophils and vascular endothelial cells(Camussi, et al., J. Exp. Med. 166:1390 (1987)).

[0008] Recent evidence associates TNF with infections (Cerami, et al.,Immunol. Today 9:28 (1988)), immune disorders, neoplastic pathologies(Oliff, et al., Cell 50:555 (1987)), autoimmune pathologies andgraft-versus host pathologies (Piguet, et al., J. Exp. Med. 166:1280(1987)). The association of TNF with cancer and infectious pathologiesis often related to the host's catabolic state. Cancer patients sufferfrom weight loss, usually associated with anorexia.

[0009] The extensive wasting which is associated with cancer, and otherdiseases, is known as “cachexia” (Kern, et al., (J. Parent. Enter. Nutr.12:286-298 (1988)). Cachexia includes progressive weight loss, anorexia,and persistent erosion of body mass in response to a malignant growth.The findamental physiological derangement can relate to a decline infood intake relative to energy expenditure. The cachectic state causesmost cancer morbidity and mortality. TNF can mediate cachexia in cancer,infectious pathology, and other catabolic states.

[0010] TNF also plays a central role in gram-negative sepsis andendotoxic shock (Michie, et al., Br. J. Surg. 76:670-671 (1989); Debets,et al., Second Vienna Shock Forum, p.463-466 (1989); Simpson, et al.,Crit. Care Clin. 5:27-47 (1989)), including fever, malaise, anorexia,and cachexia. Endotoxin strongly activates monocyte/macrophageproduction and secretion of TNF and other cytokines (Kombluth, et al.,J.Immunol. 137:2585-2591 (1986)). TNF and other monocyte-derived cytokinesmediate the metabolic and neurohormonal responses to endotoxin (Michie,et al., New. Engl. J. Med. 318:1481-1486 (1988)). Endotoxinadministration to human volunteers produces acute illness with flu-likesymptoms including fever, tachycardia, increased metabolic rate andstress hormone release (Revhaug, et al., Arch. Surg. 123:162-170(1988)). Circulating TNF increases in patients suffering fromGram-negative sepsis (Waage, et al., Lancet 1:355-357 (1987); Hammerle,et al., Second Vienna Shock Forum p. 715-718 (1989); Debets, et al.,Crit. Care Med. 17:489-497 (1989); Calandra, et al.,J. Infect. Dis.161:982-987 (1990)).

TNF Antibodies

[0011] Polyclonal murine antibodies to TNF are disclosed by Cerami etal. (EPO Patent Publication 0212489, Mar. 4, 1987). Such antibodies weresaid to be useful in diagnostic immunoassays and in therapy of shock inbacterial infections.

[0012] Rubin et al. (EPO Patent Publication 0218868, April 22, 1987)discloses murine monoclonal antibodies to human TNF, the hybridomassecreting such antibodies, methods of producing such murine antibodies,and the use of such murine antibodies in immunoassay of TNF.

[0013] Yone et al. (EPO Patent Publication 0288088, Oct. 26, 1988)discloses anti-TNF murine antibodies, including mAbs, and their utilityin immunoassay diagnosis of pathologies, in particular Kawasaki'spathology and bacterial infection. The body fluids of patients withKawasaki's pathology (infantile acute febrile mucocutaneous lymph nodesyndrome; Kawasaki, Allergy 16:178 (1967); Kawasaki, Shonica(Pediatrics) 26:935 (1985)) were said to contain elevated TNF levelswhich were related to progress of the pathology (Yone et al., infra).

[0014] Other investigators have described rodent or murine mAbs specificfor recombinant human TNF which had neutralizing activity in vitro(Liang, et al., (Biochem. Biophys. Res. Comm. 137:847-854 (1986);Meager, et al., Hybridoma 6:305-311 (1987); Fendly et al., Hybridoma6:359-369 (1987); Bringman, et al., Hybridoma 6:489-507 (1987); Hirai,et al.,J. Immunol. Meth. 96:57-62 (1987); Moller, et al., (Cytokine2:162-169 (1990)). Some of these mAbs were used to map epitopes of humanTNF and develop enzyme immunoassays (Fendly et al., infra; Hirai et al.,infra; Moller et al., infra) and to assist in the purification ofrecombinant TNF (Bringinan et al., infra). However, these studies do notprovide a basis for producing TNF neutralizing antibodies that can beused for in vivo diagnostic or therapeutic uses in humans, due toimmunogenicity, lack of specificity and/or pharmaceutical suitability.

[0015] Neutralizing antisera or mAbs to TNF have been shown in mammalsother than man to abrogate adverse physiological changes and preventdeath after lethal challenge in experimental endotoxemia and bacteremia.This effect has been demonstrated, e.g., in rodent lethality assays andin primate pathology model systems (Mathison, et al., J. Clin. Invest.81:1925-1937 (1988); Beutler, et al., Science 229:869-871 (1985);Tracey, et al., Nature 330:662-664 (1987); Shimamoto, et al., Immunol.Lett. 17:311-318 (1988); Silva, et al.,J. Infect. Dis. 162:421-427(1990); Opal,et al.,J. Infect. Dis. 161:1148-1152 (1990); Hinshaw, etal., Circ. Shock 30:279-292 (1990)).

[0016] Putative receptor binding loci of hTNF has been disclosed by Eckand Sprang (J. Biol. Chem. 264(29), 17595-17605 (1989)), who identifiedthe receptor binding loci of TNF-α as consisting of amino acids 11-13,37-42, 49-57 and 155-157.

[0017] PCT publication W091/02078 (1991) discloses TNF ligands which canbind to monoclonal antibodies having the following epitopes: at leastone of 1-20, 56-77, and 108-127; at least two of 1-20, 56-77, 108-127and 138-149; all of 1-18, 58-65, 115-125 and 138-149; all of 1-18, and108-128; all of 56-79, 110-127 and 135- or 136-155; all of 1-30, 117-128and 141-153; all of 1-26, 117-128 and 141-153; all of 22-40, 49-96 or49-97, 110-127 and 136-153; all of 12-22, 36-45, 96-105 and 132-157;both of 1-20 and 76-90; all of 22-40, 69-97, 105-128 and 135-155; all of22-31 and 146-157; all of 22-40 and 49-98; at least one of 22-40, 49-98and 69-97, both of 22-40 and 70-87.

[0018] To date, experience with anti-TNF murine mAb therapy in humanshas been limited. In a phase I study, fourteen patients with severeseptic shock were administered a murine anti-TNF mAb in a single dosefrom 0.4-10 mg/kg (Exley, A. R. et al., Lancet 335:1275-1277 (1990)).However, seven of the fourteen patients developed a human anti-murineantibody response to the treatment, which treatment suffers from theknown problems due to immunogenicity from the use of murine heavy andlight chain portions of the antibody. Such immunogenicity causesdecreased effectiveness of continued administration and can rendertreatment ineffective, in patients undergoing diagnostic or therapeuticadministration of murine anti-TNF antibodies.

[0019] Administration of murine TNF mAb to patients suffering fromsevere graft versus host pathology has also been reported (Herve, etal., Lymphoma Res. 9:591 (1990)).

TNF Receptors

[0020] The numerous biological effects of TNFα and the closely relatedcytokine, TNFβ (lymphotoxin), are mediated by two TNF transmembranereceptors, both of which have been cloned. The p55 receptor (also termedTNF-R55, TNF-RI, or TNFRβ) is a 55 kDa glycoprotein shown to transducesignals resulting in cytotoxic, anti-viral, and proliferative activitiesof TNFα.

[0021] The p75 receptor (also termed TNF-R75, TNF-RII, or TNFRα) is a 75kDa glycoprotein that has also been shown to transduce cytotoxic andproliferative signals as well as signals resulting in the secretion ofGM-CSF. The extracellular domains of the two receptors have 28% homologyand have in common a set of four subdomains defined by numerousconserved cysteine residues. The p75 receptor differs, however, byhaving a region adjacent to the transmembrane domain that is rich inproline residues and contains sites for O-linked glycosylation.Interestingly, the cytoplasmic domains of the two receptors share noapparent homology which is consistent with observations that they cantransduce different signals to the interior of the cell.

[0022] TNFα inhibiting proteins have been detected in normal human urineand in serum of patients with cancer or endotoxemia. These have sincebeen shown to be the extracellular domains of TNF receptors derived byproteolytic cleavage of the transmembrane forms. Many of the samestimuli that result in TNFα release also result in the release of thesoluble receptors, suggesting that these soluble TNFα inhibitors canserve as part of a negative feedback mechanism to control TNFα activity.

[0023] Aderka, et al., Isrl. J. Med. Sci. 28:126-130 (1992) disclosessoluble forms of TNF receptors (sTNF-Rs) which specifically bind TNF andthus can compete with cell surface TNF receptors to bind TNF (Seckinger,et al.,J. Exp. Med. 167:151 1-1516 (1988); Engelmann, et al.,J. Biol.Chem. 264:11974-11980 (1989)).

[0024] Loetscher, et al., Cell 61:351-359 (Apr. 20, 1990) discloses thecloning and expression of human 55 kd TNF receptor with the partialamino acid sequence, complete cDNA sequence and predicted amino acidsequence.

[0025] Schall et al., Cell 61:361-370 (Apr. 20, 1990), disclosesmolecular cloning and expression of a receptor for human TNF with anisolated cDNA clone including a receptor as a 415 amino acid proteinwith an apparent molecular weight of 28 kDa, as well as the cDNAsequence and predicted amino acid sequence.

[0026] Nophar, et al., EMBO J. 9(10):3269-3278 (1990) discloses solubleforms of TNF receptor and that the cDNA for type I TNF-R encodes boththe cell surface and soluble forms of the receptor. The cDNA andpredicted amino acid sequences are disclosed.

[0027] Engelmann, et al.,J. Biol. Chem. 265(3):1531-1536 (1990),discloses TNF-binding proteins, purified from human urine, both havingan approximate molecular weight of 30 kDa and binding TNF-α moreeffectively than TNF-B. Sequence data is not disclosed. See alsoEngelmann, et al.,J. Biol. Chem. 264 (20):11974-11980 (1989).

[0028] European Patent publication number 0 433 900 Al, published June26, 1991, owned by YEDA Research and Development Co., Ltd., Wallach, etal., discloses TNF binding protein I (TBP-I), derivatives and analogsthereof, produced expression of a DNA encoding the entire human type ITNF receptor, or a soluble domain thereof.

[0029] PCT publication number WO 92/13095, published Aug. 6, 1992, ownedby Synergen, Carmichael et al., discloses methods for treating tumornecrosis factor mediated diseases by administration of a therapeuticallyeffective amount of a TNF inhibitor selected from a 30 kDa TNF inhibitorand a 40 kDa TNF inhibitor selected from the full length 40 kDa TNFinhibitor or modifications thereof.

[0030] European Patent Publication number 0 526 905 A2, published Oct.2, 1993, owned by YEDA Research and Development Company, Ltd., Wallachet al., discloses multimers of the soluble forms of TNF receptorsproduced by either chemical or recombinant methods which are useful forprotecting mammals from the deleterious effects of TNF, which includeportions of the hp55 TNF-receptor.

[0031] PCT publication WO 92/07076, published Apr. 30, 1992, owned byCharring Cross Sunley Research Center, Feldmann et al., disclosesmodified human TNFα receptor which consists of the first threecysteine-rich subdomains but lacks the fourth cysteine-rich subdomain ofthe extracellular binding domain of the 55 kDa or 75 kDa TNF receptorfor human TNFα, or an amino acid sequence having a homology of 90% ormore with the TNF receptor sequences.

[0032] European Patent Publication 0 412 486 A1, published Feb. 13,1991, owned by YEDA Research and Development Co., Ltd., Wallach et al.,discloses antibodies to TNF binding protein I (TBP-I), and fragmentsthereof, which can be used as diagnostic assays or pharmaceuticalagents, either inhibiting or mimicking the effects of TNF on cells.

[0033] European Patent Publication number 0 398 327 A1, published Nov.22, 1990, owned by YEDA Research and Development Co., Ltd., Wallach etal., discloses TNF binding protein (TBP), isolated and purified, havinginhibitory activity on the cytotoxic effect of TNF, as well as TNFbinding protein II and salts, functional derivatives, precursors andactive fractions thereof, as well as polyclonal and monoclonalantibodies to TNF binding protein II.

[0034] European Patent Publication 0 308 378 A2, published Mar. 22,1989, owned by YEDA Research and Development Co., Ltd., Wallach, et al.,discloses TNF inhibitory protein isolated and substantially purified,having activity to inhibit the binding of TNF to TNF receptors and toinhibit the cytotoxicity of TNF. Additionally disclosed are TNFinhibitory protein, salts, functional derivatives and active fractionsthereof, used to antagonize the deleterious effects of TNF.

[0035] Accordingly, there is a need to provide novel TNF antibodies orpeptides which overcome the problems of murine antibody immunogenicityand which provide reduced immunogenicity and increased neutralizationactivity.

[0036] Citation of documents herein is not intended as an admission thatany of the documents cited herein is pertinent prior art, or anadmission that the cited documents is considered material to thepatentability of any of the claims of the present application. Allstatements as to the date or representation as to the contents of thesedocuments is based on the information available to the applicant anddoes not constitute any admission as to the correctness of the dates orcontents of these documents.

SUMMARY OF THE INVENTION

[0037] It is the object of the present invention to overcome one or moredeficiencies of the background art.

[0038] It is also an object of the present invention to provide methodshaving utility for in vitro, in situ and/or in vivo diagnosis and/ortreatment of animal cells, tissues or pathologies associated with thepresence of tumor necrosis factor (TNF), using anti-TNF antibodiesand/or anti-TNF peptides.

[0039] Anti-TNF antibodies (Abs) are intended to include at least one ofmonoclonal rodent-human chimeric antibodies, rodent antibodies, humanantibodies or any portions thereof, having at least one antigen bindingregion of an immunoglobulin variable region, which antibody binds TNF.

[0040] Anti-TNF peptides are capable of binding TNF under physiologicalconditions, and can include, but are not limited to, portions of a TNFreceptor and/or portions or structural analogs of anti-TNF antibodyantigen binding regions or variable regions. Such antibodies or peptidesbind TNF with neutralizing and/or inhibiting biological activity.

[0041] Anti-TNF antibodies and/or anti-TNF peptides of the presentinvention can be routinely made and/or used according to methods of thepresent invention, such as, but not limited to synthetic methods,hybridomas, and/or recombinant cells expressing nucleic acid encodingsuch anti-TNF antibodies or peptides.

[0042] The present invention also provides antigenic polypeptides ofhTNF, corresponding to peptides containing neutralizing epitopes orportions of TNF that, when such epitopes on TNF are bound by anti-TNFantibodies or peptides, neutralize or inhibit the biological activity ofTNF in vitro, in situ or in vivo.

[0043] The present invention also provides anti-TNF antibodies andpeptides in the form of pharmaceutical and/or diagnostic compoundsand/or compositions, useful for the diagnostic and/or therapeuticmethods of the present invention for diagnosing and/or treatingTNF-related pathologies.

[0044] Anti-TNF Abs or anti-TNF peptides of the present invention areprovided for use in diagnostic methods for detecting TNF in patients oranimals suspected of suffering from conditions associated with abnormalTNF production, including methods wherein high affinity anti-TNFantibodies or peptides are contacted with a biological sample from apatient and an antigen-antibody reaction detected. Also included in thepresent invention are kits for detecting TNF in a solution usinganti-TNF antibodies or peptides, preferably in detectably labeled form.

[0045] The present invention is also directed to an anti-hTNF chimericantibody comprising two light chains and two heavy chains, each of thechains comprising at least part of a human constant region and at leastpart of a variable (V) region of non-human origin having specificity tohuman TNF, said antibody binding with high affinity to a inhibitingand/or neutralizing epitope of human TNF, such as the antibody cA2. Theinvention also includes a fragments or a derivative such an antibody,such as one or more portions of the antibody chain, such as the heavychain constant, joining, diversity or variable regions, or the lightchain constant, joining or variable regions.

[0046] Methods are also provided for making and using anti-TNFantibodies and peptides for various utilities of the present invention,such as but not limited to, hybridoma, recombinant or chemical syntheticmethods for producing anti-TNF antibodies or anti-TNF peptides accordingto the present invention; detecting TNF in a solution or cell; removingTNF from a solution or cell, inhibiting one or more biologicalactivities of TNF in vitro, in situ or in vitro. Such removal caninclude treatment methods of the present invention for alleviatingsymptoms or pathologies involving TNF, such as, by not limited tobacterial, viral or parasitic infections, chronic inflammatory diseases,autoimmune diseases, malignancies, and/or neurodegenerative diseases.

[0047] The invention further relates to the unexpected discovery thatthe inhibition or antagonism of TNF decreases the expression of VascularEndothelial Growth Factor (VEGF) or Vascular Permeability Factor (VPF).VEGF has been implicated in the angiogenesis in cancer, vasculardiseases and rheumatoid arthritis, for example. Thus, a TNF antagonist,such as an anti-TNF antibody, can be administered to a mammal for thetreatment to decrease angiogenesis, such as in the treatment of aVEGF-mediated disease.

BRIEF DESCRIPTION OF THE DRAWINGS

[0048]FIG. 1 is a graph showing dose dependent binding of mouse mAb A2to human TNFα.

[0049]FIG. 2 is a graph showing lack of recognition of heat-inactivatedhuman TNFα by mAb A2.

[0050]FIG. 3 is a graph showing neutralization of in vitro TNFcytotoxicity by murine A2. Control: murine IgG1 anti-lipid A mAb (8A1)with natural human TNF. Average absorbance values for controls were asfollows: no TNF added=1.08; natural TNF, no antibody=0.290; andrecombinant TNF, no antibody=0.500.

[0051]FIG. 4 is a graph showing that mAb A2 and chimeric A2 do notinhibit or neutralize human lymphotoxin (TNFβ).

[0052]FIG. 5 is a graph showing that mAbs murine A2 and chimeric CA2 donot inhibit or neutralize murine TNFα.

[0053]FIG. 6 and FIG. 7 are graphs showing that mAb A2 inhibits orneutralizes TNF produced by chimpanzee monocytes and rhTNFα.

[0054]FIGS. 8A and 8B provide schematic diagrams of the plasmids usedfor expression of the chimeric H (pA2HG1apgpt) and L (pA2HuKapgpt)chains of the chimeric A2 antibody.

[0055]FIGS. 9A and 9B are graphs showing results of a cross-blockingepitope ELISA with murine A2 (mA2) and chimeric (cA2) antibodycompetitors.

[0056]FIG. 10A and 10B are graphs of a Scatchard analysis of¹²⁵I-labelled mAb A2 (mA2) and chimeric A2 (cA2) binding to recombinanthuman TNFα immobilized on a microtiter plate. Each Ka value wascalculated from the average of two independent determinations.

[0057]FIG. 11 is a graph showing neutralization of TNF cytotoxicity bychimeric A2. The control is a chimeric mouse/human IgG1 anti-plateletmAb (7E3) reacting with natural human TNF. Average absorbance values forcontrols were: no TNF added =1.08; natural TNF, no Ab=0.290; andrecombinant TNF, no Ab=0.500.

[0058]FIG. 12 is a graph showing in vitro neutralization of TNF-inducedELAM-1 expression by chimeric A2. The control is a chimeric mouse/humanIgG1 anti-CD4 antibody.

[0059]FIG. 13 is an amino acid sequence of human TNF as SEQ ID NO: 1.

[0060] FIGS. 14A-14B. FIG. 14A is a graphical representation of epitopemapping of chimeric mAb cA2 indicating relative binding of cA2 to humanTNF peptide pins. FIG. 14B is a graphical representation of epitopemapping of chimeric mAb cA2 indicating relative binding of cA2 to humanTNF peptide pins in the presence of human TNF.

[0061]FIG. 15 is an amino acid sequence of human TNF showing sequenceshaving portions of epitopes recognized by cA2, corresponding to portionsof amino acids 59-80 and/or 87-108 of SEQ ID NO:1.

[0062] FIGS. 16A-16B. FIG. 16A is a nucleic acid sequence (SEQ ID NO:2)and corresponding amino acid sequence (SEQ ID NO:3) of a cloned cA2light chain variable region. FIG. 16B is a nucleic acid sequence (SEQ IDNO:4) and corresponding amino acid sequence (SEQ ID NO:5) of a clonedcA2 heavy chain variable region.

[0063]FIG. 17 is a graphical representation of the early morningstiffness for the five patients in group I, and the four patients ingroup II is plotted as the mean percent of the baseline value versustime. Both groups showed an approximately 80 percent decrease or greaterin early morning stiffness, which persisted for greater than 40 days.

[0064]FIG. 18 is a graphical representation of the assessment of painusing a visual analogue scale for the five patients in group I, and thefour patients in group II, is plotted as the mean percent of thebaseline value versus time. Both groups showed an approximately 60 to 80percent decrease in pain score which persisted for greater than 40 days.

[0065]FIG. 19 is a graphical representation of the Ritchie ArticularIndex, (a scale scored of joint tenderness), plotted as the mean percentof the baseline value versus time. Both groups showed an approximately80 percent decrease in the Ritchie Articular Index, which persisted forgreater than 40 days.

[0066]FIG. 20 is a graphical representation of the number of swollenjoints for the five patients in group I and the four patients in GroupII plotted as the mean percent of baseline value versus time. Bothgroups showed an approximately 70 to 80 percent decrease in swollenjoints, which persisted for 30 to 40 days.

[0067]FIG. 21 is a graphical representation of the serum C-reactiveprotein for four to five patients in group I, and three of the fourpatients in group II, plotted as the mean percent of the baseline valueversus time. Both groups showed an approximately 80 percent reduction inCRP which persisted for 30 to 40 days. The values for patient number 1and patient number 7 were omitted from the computations on which theplots are based, since these patients did not have elevated CRP valuesat baseline.

[0068]FIG. 22 is a graphical representation of the erythrocytesedimentation rate for the five patients in group I and three of thepatients in group II plotted as the mean percent of the baseline valueversus time. Both groups showed an approximately 40 percent reduction inESR which persisted for at least 40 days. The data from patient number 9is omitted from the computations on which the plots were based, sincethis patient did not have an elevated ESR at baseline.

[0069]FIG. 23 is a graphical representation of the index of DiseaseActivity, (a composite score of several parameters of disease activity),for the five patients in group I, and the four patients in group II,plotted as the mean percent of the baseline value versus time. Bothgroups showed a clinically significant reduction in IDA, which persistedfor at least 40 days.

[0070]FIG. 24 is a graphical representation of swollen joint counts(maximum 28), as recorded by a single observer. Circles representindividual patients and horizontal bars show median values at each timepoint. The screening time point was within 4 weeks of entry to the study(week 0); data from patient 15 were not included after week 2 (dropout).Significance of the changes, relative to week 0, by Mann Whitney test,adjusted: week 1, p>0.05; week 2, p<0.02; weeks 3-4, p<0.002; weeks 6-8,p<0.001.

[0071]FIG. 25 is a graphical representation of levels of serumC—reactive protein (CRP) -Serum CRP (normal range 0-10 mg/liter),measured by nephelometry. Circles represent individual patients andhorizontal bars show median values at each time point. The screeningtime point was within 4 weeks of entry to the study (week 0); data frompatient 15 were not included after week 2 (dropout). Significance of thechanges, relative to week 0, by Mann-Whitney test, adjusted: week 1,p<0.00l; week 2, p<0.003; week 3, p<0.002; week 4, p<0.02; week 6,8,p<0.001.

[0072] FIGS. 26A-26B. FIG. 26A is a schematic illustration of the genesencoding TNF receptor/IgG fusion proteins and the gene encoding thetruncated light chain. The gene encoding Ig heavy chain (IgH) fusionproteins had the same basic structure as the naturally occurring,rearranged Ig genes except that the Ig variable region coding sequencewas replaced with TNF receptor coding sequence. Except for the TNFreceptor coding sequences and a partial human K sequence derived bymodifying the marine J region coding sequence in the cM-T412 IgH gene byPCT mutagenesis, the entire genomic fragment shown originated from thecM-T412 chimeric mouse/human IgH gene. Looney et al., Hum. AntibodyHybrid. 3:191-200 (1992). The region deleted in the genes encodingp55-sf3 and p75P-sf3 is marked in the figure. The JC_(K) gene, encodinga truncated Ig Kappa light chain, was constructed by deleting thevariable region coding sequence from the cM-T412 chimeric mouse/human IgKappa gene (Looney, infra) and using PCR mutagenesis to change themarine J sequence to a partial human J sequence. The p55-light chainfusion in p55-df2 was made by inserting the p55 coding sequence into theEcoRV site in the JCK gene. Tracey et al., Nature 330:662-666 (1987).FIG. 26B is a schematic illustration of several immunoreceptor moleculesof the present invention. The blackened ovals each represent a domain ofthe IgGG constant region. The circles represent the truncated lightchain. Small circles adjacent to a p55 or p75 subunit mark the positionsof human 3 sequence. The incomplete circles in p75-sf2 and -sf3 are toillustrate that the C-terminal 53 amino acids of the p75 extracellulardomain were deleted. Lines between subunits represent disulfide bonds.

[0073]FIG. 27 is a schematic illustration of the construction of acM-T412 heavy chain so that it has a unique cloning site for insertionof foreign genes such as p55 and p75.

[0074]FIG. 28 is a schematic illustration of the construction of thevectors used to express the heavy chain of the immunoreceptors.

[0075]FIG. 29 is a schematic illustration of the construction of acM-T412 light chain so that it has a unique cloning site for insertionof foreign genes such as p55 and p75.

[0076]FIG. 30 is a schematic illustration of the construction of thevectors used to express the light chain of the immunoreceptors.

[0077] FIGS. 31A-31C are graphical representations showing that fusionproteins protected WEHI 164 cells from TNFα cytotoxicity. Cells werefirst sensitized to TNFα with actinomycin D and then incubated in 2ng/ml TNFα with varying concentrations of TNFα overnight at 37° C. Cellviability was determined by measuring their uptake of MTT dye. FIG. 31Ashows p55 fusion proteins. FIG. 31B shows p75 fusion proteins. FIG. 31Cshows comparison of the protective ability of the non-fusion form of p55(p55-nf) to p55-sf2.

[0078]FIG. 32 is a graphical representation of data showing that fusionproteins also effectively protect WEHI 164 cells from TNFβ cytotoxicity.

[0079] FIGS. 33A-33H are graphical representations of analyses ofbinding between the various fusion proteins and TNFα by saturationbinding (FIG. 33A and 33B) and Scatchard analysis (FIG. 33C-33H). Amicrotiter plate was coated with excess goat anti-Fc polyclonal antibodyand incubated with 10 ng/ml of fusion protein in TBST buffer (10 mMTris-HCl, pH 7.8, 150 mM NaCl, 0.05% Tween-20) for 1 hour. Varyingamounts of ¹²⁵I labeled TNFα (specific activity—34.8 μCi/μg) were thenincubated with the captured fusion protein in PBS (10 mM Na Phosphate,pH 7.0, 150 mM NaCl) with 1% bovine serum albumin for 2 hours. UnboundTNFα was washed away with four washes in PBS and the cpm bound wasquantitated using a y-counter. All samples were analyzed in triplicate.The slope of the lines in (FIGS. 33C-H) represent the affinity constant,K_(a). The dissociation constant (K_(d)) values (see Table 1) werederived using the equation K_(d)=I/K_(a).

[0080]FIG. 34 is a graphic illustration depicting VEGF levels in theserum of rheumatoid arthritis patients treated with placebo (circles), 1mg/kg cA2 antibody (square) or 10 mg/kg (triangle). The figure showsthat the administration of an anti-TNF antibody resulted in decreasedlevels of VEGF.

DETAILED DESCRIPTION OF THE INVENTION

[0081] Tumor necrosis factor (TNF) has been discovered to mediate or beinvolved in many pathologies, such as, but not limited to, bacterial,viral or parasitic infections, chronic inflammatory diseases, autoimmunediseases, malignancies, and/or neurodegenerative diseases. Accordingly,anti-TNF compounds and compositions of the present invention which haveneutralizing and/or inhibiting activity against TNF are discovered toprovide methods for treating and/or diagnosing such pathologies.

[0082] The present invention thus provides anti-TNF compounds andcompositions comprising anti-TNF antibodies (Abs) and/or anti-TNFpeptides which inhibit and/or neutralize TNF biological activity invitro, in situ and/or in vivo, as specific for association withneutralizing epitopes of human tumor necrosis factor-alpha (hTNFα)and/or human tumor necrosis factor β (hTNFβ). Such anti-TNF Abs orpeptides have utilities for use in research, diagnostic and/ortherapeutic methods of the present invention for diagnosing and/ortreating animals or humans having pathologies or conditions associatedwith the presence of a substance reactive with an anti-TNF antibody,such as TNF or metabolic products thereof. Such pathologies can includethe generalized or local presence of TNF or related compounds, inamounts and/or concentrations exceeding, or less than, those present ina normal healthy subject, or as related to a pathological condition.

Anti-TNF Antibodies and Methods

[0083] The term “antibody” is meant to include polyclonal antibodies,monoclonal antibodies (mAbs), chimeric antibodies, anti-idiotypic(anti-Id) antibodies to antibodies that can be labeled in soluble orbound form, as well as fragments, regions or derivatives thereof,provided by any known technique, such as, but not limited to, enzymaticcleavage, peptide synthesis or recombinant techniques. Such anti-TNFantibodies of the present invention are capable of binding portions ofTNF that inhibit the binding of TNF to TNF receptors.

[0084] Polyclonal antibodies are heterogeneous populations of antibodymolecules derived from the sera of animals immunized with an antigen. Amonoclonal antibody contains a substantially homogeneous population ofantibodies specific to antigens, which population contains substantiallysimilar epitope binding sites. MAbs may be obtained by methods known tothose skilled in the art. See, for example Kohler and Milstein, Nature256:495-497 (1975); U.S. Pat. No. 4,376,110; Ausubel et al., eds.,Current Protocols in Molecular Biology, Greene Publishing Assoc. andWiley Interscience, N.Y., (1987, 1992); and Harlow and Lane ANTIBODIES:A Laboratory Manual Cold Spring Harbor Laboratory (1988); Colligan etal., eds., Current Protocols in Immunology, Greene Publishing Assoc. andWiley Interscience, N.Y., (1992, 1993), the contents of which referencesare incorporated entirely herein by reference. Such antibodies may be ofany immunoglobulin class including IgG, IgM, IgE, IgA, GILD and anysubclass thereof. A hybridoma producing a mAb of the present inventionmay be cultivated in vitro, in situ or in vivo. Production of hightiters of mAbs in vivo or in situ makes this the presently preferredmethod of production.

[0085] Chimeric antibodies are molecules different portions of which arederived from different animal species, such as those having variableregion derived from a murine mAb and a human immunoglobulin constantregion, which are primarily used to reduce inimunogenicity inapplication and to increase yields in production, for example, wheremurine mAbs have higher yields from hybridomas but higher immunogenicityin humans, such that human/murine chimeric mAbs are used. Chimericantibodies and methods for their production are known in the art(Cabilly et al., Proc. Natl. Acad. Sci. USA 81:3273-3277 (1984);Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984);Boulianne et al., Nature 312:643-646 (1984); Cabilly et al., EuropeanPatent Application 125023 (published Nov. 14, 1984); Neuberger et al.,Nature 314:268-270 (1985); Taniguchi et al., European Patent Application171496 (published Feb. 19, 1985); Morrison et al., European PatentApplication 173494 (published Mar. 5, 1986); Neuberger et al., PCTApplication WO 86/01533, (published Mar. 13, 1986); Kudo et al.,European Patent Application 184187 (published Jun. 11, 1986); Morrisonet al., European Patent Application 173494 (published Mar. 5, 1986);Sahagan et al.,J. Immunol. 137:1066-1074 (1986); Robinson et al.,International Patent Publication #PCT/US86/02269 (published May 7,1987); Liu et al., Proc. Natl. Acad. Sci. USA 84:3439-3443 (1987); Sunet al., Proc. Natl. Acad. Sci. USA 84:214-218 (1987); Better et al.,Science 240:1041-1043 (1988); and Harlow and Lane Antibodies: aLaboratory Manual Cold Spring Harbor Laboratory (1988)). Thesereferences are entirely incorporated herein by reference.

[0086] An anti-idiotypic (anti-Id) antibody is an antibody whichrecognizes unique determinants generally associated with theantigen-binding site of an antibody. An Id antibody can be prepared byimmunizing an animal of the same species and genetic type (e.g., mousestrain) as the source of the mAb with the mAb to which an anti-Id isbeing prepared. The immunized animal will recognize and respond to theidiotypic determinants of the immunizing antibody by producing anantibody to these idiotypic determinants (the anti-Id antibody). See,for example, U.S. Pat. No. 4,699,880, which is herein entirelyincorporated by reference.

[0087] The anti-Id antibody may also be used as an “immunogen” to inducean immune response in yet another animal, producing a so-calledanti-anti-Id antibody. The anti-anti-Id may be epitopically identical tothe original mAb which induced the anti-Id. Thus, by using antibodies tothe idiotypic determinants of a mAb, it is possible to identify otherclones expressing antibodies of identical specificity.

[0088] Anti-TNF antibodies of the present invention can include at leastone of a heavy chain constant region (H_(c)), a heavy chain variableregion (H_(v)), a light chain variable region (L_(v)) and a light chainconstant region (L_(c)), wherein a polyclonal Ab, monoclonal Ab,fragment and/or regions thereof include at least one heavy chainvariable region (H_(v)) or light chain variable region (L_(v)) whichbinds a portion of a TNF and inhibits and/or neutralizes at least oneTNF biological activity.

[0089] Preferred antibodies of the present invention are high affinityhuman-murine chimeric anti-TNF antibodies, and fragments or regionsthereof, that have potent inhibiting and/or neutralizing activity invivo against human TNFα. Such antibodies and chimeric antibodies caninclude those generated by immunization using purified recombinant hTNFα(SEQ ID NO: 1) or peptide fragments thereof. Such fragments can includeepitopes of at least 5 amino acids of residues 87-108, or a combinationof both of 59-80 and 87-108 of hTNFα (as these corresponding amino acidsof SEQ ID NO:1). Additionally, preferred antibodies, fragments andregions of anti-TNF antibodies of the present invention do not recognizeamino acids from at least one of amino acids 11 - 13, 37-42, 49-57 or155-157 of hTNFα (of SEQ ID NO: 1).

[0090] Preferred anti-TNF mAbs are also those which will competitivelyinhibit in vivo the binding to human TNFα of anti-TNFα murine mAb A2,chimeric mAb cA2, or an antibody having substantially the same specificbinding characteristics, as well as fragments and regions thereof.Preferred antibodies of the present invention are those that bindepitopes recognized by A2 and cA2, which are included in amino acids59-80 and/or 87-108 of hTNFα (as these corresponding amino acids of SEQID NO:1), such that the epitopes consist of at least 5 amino acids whichcomprise at least one amino acid from the above portions of human TNFα.

[0091] Preferred methods for determining mAb specificity and affinity bycompetitive inhibition can be found in Harlow, et al., Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, NY, 1988), Colligan et al., eds., Current Protocols inImmunology, Greene Publishing Assoc. and Wiley Interscience, N.Y.,(1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), whichreferences are entirely incorporated herein by reference.

[0092] The techniques to raise antibodies of the present invention tosmall peptide sequences that recognize and bind to those sequences inthe free or conjugated form or when presented as a native sequence inthe context of a large protein are well known in the art. Suchantibodies include murine, murine-human and human-human antibodiesproduced by hybridoma or recombinant techniques known in the art.

[0093] As used herein, the term “antigen binding region” refers to thatportion of an antibody molecule which contains the amino acid residuesthat interact with an antigen and confer on the antibody its specificityand affinity for the antigen. The antibody region includes the“framework” amino acid residues necessary to maintain the properconformation of the antigen-binding residues.

[0094] Preferably, the antigen binding region will be of murine origin.In other embodiments, the antigen binding region can be derived fromother animal species, in particular rodents such as rabbit, rat orhamster.

[0095] The antigen binding region of the chimeric antibody of thepresent invention is preferably derived from a non-human antibodyspecific for human TNF. Preferred sources for the DNA encoding such anon-human antibody include cell lines which produce antibody, preferablyhybrid cell lines commonly known as hybridomas. A preferred hybridoma isthe A2 hybridoma cell line.

[0096] An “antigen” is a molecule or a portion of a molecule capable ofbeing bound by an antibody which is additionally capable of inducing ananimal to produce antibody capable of binding to an epitope of thatantigen. An antigen can have one or more than one epitope. The specificreaction referred to above is meant to indicate that the antigen willreact, in a highly selective manner, with its corresponding antibody andnot with the multitude of other antibodies which can be evoked by otherantigens. Preferred antigens that bind antibodies, fragments and regionsof anti-TNF antibodies of the present invention include at least 5 aminoacids comprising at least one of amino acids residues 87-108 or bothresidues 59-80 and 87-108 of hTNFα (of SEQ ID NO:1). Preferred antigensthat bind antibodies, fragments and regions of anti-TNF antibodies ofthe present invention do not include amino acids of amino acids 11-13,37-42, 49-57 or 155-157 of hTNFα (SEQ ID NO:1).

[0097] Particular peptides which can be used to generate antibodies ofthe present invention can include combinations of amino acids selectedfrom at least residues 87-108 or both residues 59-80 and 87-108, whichare combined to provide an epitope of TNF that is bound by anti-TNFantibodies, fragments and regions thereof, and which binding providedanti-TNF biological activity. Such epitopes include at least 1-5 aminoacids and less than 22 amino acids from residues 87-108 or each ofresidues 59-80 and 87-108, which in combination with other amino acidsof TNF provide epitopes of at least 5 amino acids in length.

[0098] TNF residues 87-108 or both residues 59-80 and 87-108 of TNF (ofSEQ ID NO: 1), fragments or combinations of peptides containing thereinare useful as immunogens to raise antibodies that will recognize peptidesequences presented in the context of the native TNF molecule.

[0099] The term “epitope” is meant to refer to that portion of anymolecule capable of being recognized by and bound by an antibody at oneor more of the Ab's antigen binding regions. Epitopes usually consist ofchemically active surface groupings of molecules such as amino acids orsugar side chains and have specific three dimensional structuralcharacteristics as well as specific charge characteristics. By“inhibiting and/or neutralizing epitope” is intended an epitope, which,when bound by an antibody, results in loss of biological activity of themolecule or organism containing the epitope, in vivo, in vitro or insitu, more preferably in vivo, including binding of TNF to a TNFreceptor.

[0100] Epitopes recognized by antibodies, and fragments and regionsthereof, of the present invention can include 5 or more amino acidscomprising at least one amino acid of each or both of the followingamino acid sequences of TNF, which provide a topographical or threedimensional epitope of TNF which is recognized by, and/or binds withanti-TNF activity, an antibody, and fragments, and variable regionsthereof, of the present invention:

[0101] 59-80:Tyr—Ser—Gln—Val—Leu—Phe—Lys—Gly—Gln—Gly—Cys—Pro—Ser—Thr—His—Val—Leu—Leu—Thr—His—Thr—Ile(AA 59-80 of SEQ ID NO: 1); and

[0102] 87-108:Tyr—Gln—Thr—Lys—Val—Asn—Leu—Leu—Ser—Ala—Ile—Lys—Ser—Pro—Cys—Gln—Arg—Glu—Thr—Pro—Glu—Gly(AA 87-108 of SEQ ID NO:1).

[0103] Preferred antibodies, fragments and regions of anti-TNFantibodies of the present invention recognize epitopes including 5 aminoacids comprising at least one amino acid from amino acids residues87-108 or both residues 59-80 and 87-108 of hTNFα (of SEQ ID NO:1).Preferred antibodies, fragments and regions of anti-TNF antibodies ofthe present invention do not recognize epitopes from at least one ofamino acids 11-13,37-42,49-57 or 155-157 of hTNFα (of SEQ ID NO:1). Inapreferred embodiment, the epitope comprises at least 2 amino acids fromresidues 87-108 or both residues 59-80 and 87-108 of hTNFα (of SEQ IDNO: 1). In another preferred embodiment, the epitope comprises at least3 amino acids from residues 59-80 and 87-108 of hTNFα (of SEQ ID NO: 1).In another preferred embodiment, the epitope comprises at least 4 aminoacids from residues 87-108 or both residues 59-80 and 87-108 of hTNFα(of SEQ ID NO: 1). In another preferred embodiment, the epitopecomprises at least 5 amino acids from residues 87-108 or both residues59-80 and 87-108 of hTNFα (of SEQ ID NO: 1). In another preferredembodiment, the epitope comprises at least 6 amino acids from residues87-108 or both residues 59-80 and 87-108 of hTNFα (of SEQ ID NO: 1). Inanother preferred embodiment, the epitope comprises at least 7 aminoacids from residues 87-108 or both residues 59-80 and 87-108 of hTNFα(of SEQ ID NO:1).

[0104] As used herein, the term “chimeric antibody” includes monovalent,divalent or polyvalent immunoglobulins. A monovalent chimeric antibodyis a dimer (HL) formed by a chimeric H chain associated throughdisulfide bridges with a chimeric L chain. A divalent chieric antibodyis tetramer (H₂L₂) formed by two HL dimers associated through at leastone disulfide bridge. A polyvalent chimeric antibody can also beproduced, for example, by employing a CH region that aggregates (e.g.,from an IgM H chain, or μ chain).

[0105] Murine and chimeric antibodies, fragments and regions of thepresent invention comprise individual heavy (H) and/or light (L)immunoglobulin chains. A chimeric H chain comprises an antigen bindingregion derived from the H chain of a non-human antibody specific forTNF, which is linked to at least a portion of a human H chain C region(CH), such as CH₁ or CH₂.

[0106] A chimeric L chain according to the present invention, comprisesan antigen binding region derived from the L chain of a non-humanantibody specific for TNF, linked to at least a portion of a human Lchain C region (CL).

[0107] Antibodies, fragments or derivatives having chimeric H chains andL chains of the same or different variable region binding specificity,can also be prepared by appropriate association of the individualpolypeptide chains, according to known method steps, e.g., according toAusubel infra, Harlow infra, and Colligan infra, the contents of whichreferences are incoporated entirely herein by reference.

[0108] With this approach, hosts expressing chimeric H chains (or theirderivatives) are separately cultured from hosts expressing chimeric Lchains (or their derivatives), and the immunoglobulin chains areseparately recovered and then associated. Alternatively, the hosts canbe co-cultured and the chains allowed to associate spontaneously in theculture medium, followed by recovery of the assembled immunoglobulin,fragment or derivative.

[0109] The hybrid cells are formed by the fusion of a non-humananti-hTNFα antibody-producing cell, typically a spleen cell of an animalimmunized against either natural or recombinant human TNF, or a peptidefragment of the human TNFα protein sequence. Alternatively, thenon-human anti-TNFα antibody-producing cell can be a B lymphocyteobtained from the blood, spleen, lymph nodes or other tissue of ananimal immunized with TNF.

[0110] The second fusion partner, which provides the immortalizingfunction, can be a lymphoblastoid cell or a plasmacytoma or myelomacell, which is not itself an antibody producing cell, but is malignant.Preferred fusion partner cells include the hybridoma SP2/0-Ag14,abbreviated as SP2/0 (ATCC CRL1581) and the myeloma P3X63Ag8 (ATCCTIB9), or its derivatives. See, e.g, Ausubel infra, Harlow infra, andColligan infra, the contents of which references are incoporatedentirely herein by reference.

[0111] Murine hybridomas which produce mAb specific for human TNFα orTNFβ are formed by the fusion of a mouse fusion partner cell, such asSP2/0, and spleen cells from mice immunized against purified hTNFα,recombinant hTNFα, natural or synthetic TNF peptides, including peptidesincluding 5 or more amino acids selected from residues 59-80, and 87-108of TNF (of SEQ ID NO: 1) or other biological preparations containingTNF. To immunize the mice, a variety of different conventional protocolscan be followed. For example, mice can receive primary and boostingimmunizations of TNF.

[0112] The antibody-producing cell contributing the nucleotide sequencesencoding the antigen-binding region of the chimeric antibody of thepresent invention can also be produced by transformation of a non-human,such as a primate, or a human cell. For example, a B lymphocyte whichproduces anti-TNF antibody can be infected and transformed with a virussuch as Epstein-Barr virus to yield an immortal anti-TNF producing cell(Kozbor et al., Immunol. Today 4:72-79 (1983)). Alternatively, the Blyrnphocyte can be transformed by providing a transforming gene ortransforming gene product, as is well-known in the art. See, e.g,Ausubel infra, Harlow infra, and Colligan infra, the contents of whichreferences are incorporated entirely herein by reference.

Antibody Production Using Hybridomas

[0113] The cell fusions are accomplished by standard procedures wellknown to those skilled in the field of immunology. Fusion partner celllines and methods for fusing and selecting hybridomas and screening formAbs are well known in the art. See, e.g, Ausubel infra, Harlow infra,and Colligan infra, the contents of which references are incoporatedentirely herein by reference.

[0114] The hTNFα-specific murine or chimeric mAb of the presentinvention can be produced in large quantities by injecting hybridoma ortransfectoma cells secreting the antibody into the peritoneal cavity ofmice and, after appropriate time, harvesting the ascites fluid whichcontains a high titer of the mAb, and isolating the mAb therefrom. Forsuch in vivo production of the mAb with a non-murine hybridoma (e.g.,rat or human), hybridoma cells are preferably grown in irradiated orathymic nude mice. Alternatively, the antibodies can be produced byculturing hybridoma or transfectoma cells in vitro and isolatingsecreted mAb from the cell culture medium or recombinantly, ineukaryotic or prokaryotic cells.

[0115] In a preferred embodiment, the antibody is a MAb which bindsamino acids of an epitope of TNF, which antibody is designated A2, rA2or cA2, which is produced by a hybridoma or by a recombinant host. Inanother preferred embodiment, the antibody is a chimeric antibody whichrecognizes an epitope recognized by A2. In a more preferred embodiment,the antibody is a chimeric antibody designated as chimeric A2 (cA2).

[0116] As examples of antibodies according to the present invention,murine mAb A2 of the present invention is produced by a cell linedesignated c134A. Chimeric antibody cA2 is produced by a cell linedesignated c168A. Cell line c134A is deposited as a research cell bankin the Centocor Cell Biology Services Depository, and cell linec168A(RCB) is deposited as a research cell bank in the CentocorCorporate Cell Culture Research and Development Depository, both atCentocor, 200 Great Valley Parkway, Malvern, Pa., 19355. The c168A cellline is also deposited at Centocor BV, Leiden, The Netherlands.

[0117] The invention also provides for “derivatives” of the murine orchimeric antibodies, fragments, regions or derivatives thereof, whichterm includes those proteins encoded by truncated or modified genes toyield molecular species functionally resembling the immunoglobulinfragments. The modifications include, but are not limited to, additionof genetic sequences coding for cytotoxic proteins such as plant andbacterial toxins. The fragments and derivatives can be produced from anyof the hosts of this invention. Alternatively, anti-TNF antibodies,fragments and regions can be bound to cytotoxic proteins or compounds invitro, to provide cytotoxic anti-TNF antibodies which would selectivelykill cells having TNF receptors.

[0118] Fragments include, for example, Fab, Fab′, F(ab′)₂ and Fv. Thesefragments lack the Fc fragment of intact antibody, clear more rapidlyfrom the circulation, and can have less non-specific tissue binding thanan intact antibody (Wahl et at.,J. NucL. Med. 24:316-325 (1983)). Thesefragments are produced from intact antibodies using methods well knownin the art, for example by proteolytic cleavage with enzymes such aspapain (to produce Fab fragments) or pepsin (to produce F(ab′)₂fragments).

[0119] The identification of these antigen binding region and/orepitopes recognized by mAbs of the present invention provides theinformation necessary to generate additional monoclonal antibodies withsimilar binding characteristics and therapeutic or diagnostic utilitythat parallel the embodiments of this application.

[0120] In a preferred embodiment, the amino acids of the epitope are notof at least one of amino acids 11-13, 37-42, 49-57 and 155-157 of hTNFα(of SEQ ID NO: 1).

[0121] Unexpectedly, anti-TNF antibodies or peptides of the presentinvention can block the action of TNF-α without binding to the putativereceptor binding locus such as is presented by Eck and Sprang (J. Biol.Chem. 264(29): 17595-17605 (1989), as amino acids 11-13, 37-42, 49-57and 155-157 of hTNFα (of SEQ ID NO:1).

Recombinant Expression of Anti-TNF Antibodies

[0122] Recombinant murine or chimeric murine-human or human-humanantibodies that inhibit TNF and bind an epitope included in the aminoacid sequences residues 87-108 or both residues 59-80 and 87-108 ofhTNFα (of SEQ ID NO: 1), can be provided according to the presentinvention using known techniques based on the teaching provided herein.See, e.g., Ausubel et al., eds. Current Protocols in Molecular Biology,Wiley Interscience, N.Y. (1987, 1992, 1993); and Sambrook et al.Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress (1989), the entire contents of which are incorporated herein byreference.

[0123] The DNA encoding an anti-TNF antibody of the present inventioncan be genomic DNA or cDNA which encodes at least one of the heavy chainconstant region (H_(c)), the heavy chain variable region (H_(v)), thelight chain variable region (L_(v)) and the light chain constant regions(L_(c)). A convenient alternative to the use of chromosomal genefragments as the source of DNA encoding the murine V regionantigen-binding segment is the use of cDNA for the construction ofchimeric immunoglobulin genes, e.g., as reported by Liu et al. (Proc.Natl. Acad. Sci., USA 84:3439 (1987) and J. Immunology 139:3521 (1987),which references are hereby entirely incorporated herein by reference.The use of cDNA requires that gene expression elements appropriate forthe host cell be combined with the gene in order to achieve synthesis ofthe desired protein. The use of cDNA sequences is advantageous overgenomic sequences (which contain introns), in that cDNA sequences can beexpressed in bacteria or other hosts which lack appropriate RNA splicingsystems.

[0124] For example, a cDNA encoding a murine V region antigen-bindingsegment having anti-TNF activity can be provided using known methodsbased on the use of the DNA sequence presented in FIG. 16A (SEQ IDNO:2). Alternatively, a cDNA encoding a murine C region antigen-bindingsegment having anti-TNF activity can be provided using known methodsbased on the use of the DNA sequence presented in FIG. 16B (SEQ IDNO:3). Probes that bind a portion of the DNA sequence presented in FIG.16A or 16B can be used to isolate DNA from hybridomas expressing TNFantibodies, fragments or regions, as presented herein, according to thepresent invention, by known methods.

[0125] Oligonucleotides representing a portion of the variable regionpresented in FIG. 16A or 16B sequence are useful for screening for thepresence of homologous genes and for the cloning of such genes encodingvariable or constant regions of an anti-TNF antibody. Such probespreferably bind to portions of sequences according to FIGS. 16A or 16Bwhich encode light chain or heavy chain variable regions which bind anactivity inhibiting epitope of TNF, especially an epitope of at least 5amino acids of residues 87-108 or a combination of residues 59-80 and87-108 (of SEQ ID NO: 1).

[0126] Such techniques for synthesizing such oligonucleotides are wellknown and disclosed by, for example, Wu, et al., Prog. Nucl. Acid. Res.Molec. Biol. 21:101-141 (1978)), and Ausubel et al., eds. CurrentProtocols in Molecular Biology, Wiley Interscience (1987, 1993), theentire contents of which are herein incorporated by reference.

[0127] Because the genetic code is degenerate, more than one codon canbe used to encode a particular amino acid (Watson, et al., infra). Usingthe genetic code, one or more different oligonucleotides can beidentified, each of which would be capable of encoding the amino acid.The probability that a particular oligonucleotide will, in fact,constitute the actual XXX-encoding sequence can be estimated byconsidering abnormal base pairing relationships and the frequency withwhich a particular codon is actually used (to encode a particular aminoacid) in eukaryotic or prokaryotic cells expressing an anti-TNF antibodyor fragment. Such “codon usage rules” are disclosed by Lathe, et al., J.Molec. Biol. 183:1-12 (1985). Using the “codon usage rules” of Lathe, asingle oligonucleotide, or a set of oligonucleotides, that contains atheoretical “most probable” nucleotide sequence capable of encodinganti-TNF variable or constant region sequences is identified.

[0128] Although occasionally an amino acid sequence can be encoded byonly a single oligonucleotide, frequently the amino acid sequence can beencoded by any of a set of similar oligonucleotides. Importantly,whereas all of the members of this set contain oligonucleotides whichare capable of encoding the peptide fragment and, thus, potentiallycontain the same oligonucleotide sequence as the gene which encodes thepeptide fragment, only one member of the set contains the nucleotidesequence that is identical to the nucleotide sequence of the gene.Because this member is present within the set, and is capable ofhybridizing to DNA even in the presence of the other members of the set,it is possible to employ the unfractionated set of oligonucleotides inthe same manner in which one would employ a single oligonucleotide toclone the gene that encodes the protein.

[0129] The oligonucleotide, or set of oligonucleotides, containing thetheoretical “most probable” sequence capable of encoding an anti-TNFantibody or fragment including a variable or constant region is used toidentify the sequence of a complementary oligonucleotide or set ofoligonucleotides which is capable of hybridizing to the “most probable”sequence, or set of sequences. An oligonucleotide containing such acomplementary sequence can be employed as a probe to identify andisolate the variable or constant region anti-TNF gene (Sambrook et al.,infra).

[0130] A suitable oligonucleotide, or set of oligonucleotides, which iscapable of encoding a fragment of the variable or constant anti-TNFregion (or which is complementary to such an oligonucleotide, or set ofoligonucleotides) is identified (using the above-described procedure),synthesized, and hybridized by means well known in the art, against aDNA or, more preferably, a cDNA preparation derived from cells which arecapable of expressing anti-TNF antibodies or variable or constantregions thereof. Single stranded oligonucleotide molecules complementaryto the “most probable” variable or constant anti-TNF region peptidecoding sequences can be synthesized using procedures which are wellknown to those of ordinary skill in the art (Belagaje, et al.,J. Biol.Chem. 254:5765-5780 (1979); Maniatis, et al., In: Molecular Mechanismsin the Control of Gene Expression, Nierlich, et al., Eds., Acad. Press,NY (1976); Wu, et al., Prog. Nucl. Acid Res. Molec. Biol. 21:101-141(1978); Khorana, Science 203:614-625 (1979)). Additionally, DNAsynthesis can be achieved through the use of automated synthesizers.Techniques of nucleic acid hybridization are disclosed by Sambrook etal. (infra), and by Hayrnes, et al. (In: Nucleic Acid Hybridization, APractical Approach, IRL Press, Washington, DC (1985)), which referencesare herein incorporated by reference. Techniques such as, or similar to,those described above have successfully enabled the cloning of genes forhuman aldehyde dehydrogenases (Hsu, et al., Proc. Natl. Acad. Sci. USA82:3771-3775 (1985)), fibronectin (Suzuki, et al., Bur. Mol. Biol.Organ. J. 4:2519-2524 (1985)), the human estrogen receptor gene (Walter,et al., Proc. Natl. Acad. Sci. USA 82:7889-7893 (1985)), tissue-typeplasminogen activator (Pennica, et al., Nature 301:214-221 (1983)) andhuman term placental alkaline phosphatase complementary DNA (Keun, etal., Proc. Natl. Acad. Sci. USA 82:8715-8719 (1985)).

[0131] In an alternative way of cloning a polynucleotide encoding ananti-TNF variable or constant region, a library of expression vectors isprepared by cloning DNA or, more preferably, cDNA (from a cell capableof expressing an anti-TNF antibody or variable or constant region) intoan expression vector. The library is then screened for members capableof expressing a protein which competitively inhibits the binding of ananti-TNF antibody, such as A2 or cA2, and which has a nucleotidesequence that is capable of encoding polypeptides that have the sameamino acid sequence as anti-TNF antibodies or fragments thereof. In thisembodiment, DNA, or more preferably cDNA, is extracted and purified froma cell which is capable of expressing an anti-TNF antibody or fragment.The purified cDNA is fragmentized (by shearing, endonuclease digestion,etc.) to produce a pool of DNA or cDNA fragments. DNA or cDNA fragmentsfrom this pool are then cloned into an expression vector in order toproduce a genomic library of expression vectors whose members eachcontain a unique cloned DNA or cDNA fragment such as in a lambda phagelibrary, expression in prokaryotic cell (e.g., bacteria) or eukaryoticcells, (e.g., mammalian, yeast, insect or, flngus). See, e.g., Ausubel,infra, Harlow, infra, Colligan, infra; Nyyssonen et al. Bio/Technology11:591-595 (Can 1993); Marks et al., Bio/Technology 11:1145-1149 (Oct.1993). Once nucleic acid encoding such variable or constant anti-TNFregions is isolated, the nucleic acid can be appropriately expressed ina host cell, along with other constant or variable heavy or light chainencoding nucleic acid, in order to provide recombinant MAbs that bindTNF with inhibitory activity. Such antibodies preferably include amurine or human anti-TNF variable region which contains a frameworkresidue having complimentarity determining residues which areresponsible for antigen binding. In a preferred embodiment, an anti-TNFvariable light or heavy chain encoded by a nucleic acid as describedabove binds an epitope of at least 5 amino acids including residues87-108 or a combination of residues 59-80 and 87-108 of hTNF (of SEQ IDNO: 1).

[0132] Human genes which encode the constant (C) regions of the murineand chimeric antibodies, fragments and regions of the present inventioncan be derived from a human fet al liver library, by known methods.Human C regions genes can be derived from any human cell including thosewhich express and produce human immunoglobulins. The human C.region canbe derived from any of the known classes or isotypes of human H chains,including gamma, μ, α, δor ε, and subtypes thereof, such as G1, G2, G3and G4. Since the H chain isotype is responsible for the variouseffector functions of an antibody, the choice of C_(H) region will beguided by the desired effector functions, such as complement fixation,or activity in antibody-dependent cellular cytotoxicity (ADCC).Preferably, the CHregion is derived from gamma 1 (IgG1 ), gamma 3(IgG3), gamma 4 (IgG4), or μ (IgM).

[0133] The human C_(L) region can be derived from either human L chainisotype, kappa or lambda.

[0134] Genes encoding human immunoglobulin C regions are obtained fromhuman cells by standard cloning techniques (Sambrook, et al. (MolecularCloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press,Cold Spring Harbor, NY (1989) and Ausubel et al., eds. Current Protocolsin Molecular Biology (1987-1993)). Human C region genes are readilyavailable from known clones containing genes representing the twoclasses of L chains, the five classes of H chains and subclassesthereof. Chimeric antibody fragments, such as F(ab′)₂ and Fab, can beprepared by designing a chimeric H chain gene which is appropriatelytruncated. For example, a chimeric gene encoding an H chain portion ofan F(ab′)2 fragment would include DNA sequences encoding the CH, domainand hinge region of the H chain, followed by a translational stop codonto yield the truncated molecule.

[0135] Generally, the murine, human or murine and chimeric antibodies,fragments and regions of the present invention are produced by cloningDNA segments encoding the H and L chain antigen-binding regions of aTNF-specific antibody, and joining these DNA segments to DNA segmentsencoding C_(H) and C_(L) regions, respectively, to produce murine, humanor chimeric immunoglobulin-encoding genes.

[0136] Thus, in a preferred embodiment, a fused chimeric gene is createdwhich comprises a first DNA segment that encodes at least theantigen-binding region of non-human origin, such as a functionallyrearranged V region with joining (J) segment, linked to a second DNAsegment encoding at least a part of a human C region.

[0137] Therefore, cDNA encoding the antibody V and C regions, the methodof producing the chimeric antibody according to the present inventioninvolves several steps, outlined below:

[0138] 1. isolation of messenger RNA (MRNA) from the cell line producingan anti-TNF antibody and from optional additional antibodies supplyingheavy and light constant regions; cloning and cDNA production therefrom;

[0139] 2. preparation of a full length cDNA library from purified mRNAfrom which the appropriate V and/or C region gene segments of the L andH chain genes can be: (i) identified with appropriate probes, (ii)sequenced, and (iii) made compatible with a C or V gene segment fromanother antibody for a chimeric antibody;

[0140] 3. Construction of complete H or L chain coding sequences bylinkage of the cloned specific V region gene segments to cloned C regiongene, as described above;

[0141] 4. Expression and production of L and H chains in selected hosts,including prokaryotic and eukaryotic cells to provide murine-murine,human-murine, human-human or human murine antibodies.

[0142] One common feature of all immunoglobulin H and L chain genes andtheir encoded mRNAs is theJ region. H and L chain J regions havedifferent sequences, but a high degree of sequence homology exists(greater than 80%) among each group, especially near the C region. Thishomology is exploited in this method and consensus sequences of H and Lchain J regions can be used to design oligonucleotides for use asprimers for introducing useful restriction sites into theJ region forsubsequent linkage of V region segments to human C region segments.

[0143] C region cDNA vectors prepared from human cells can be modifiedby site-directed mutagenesis to place a restriction site at theanalogous position in the human sequence. For example, one can clone thecomplete human kappa chain C (C_(k)) region and the complete humangamma-1 C region (C_(gamma-1)). In this case, the alternative methodbased upon genomic C region clones as the source for C region vectorswould not allow these genes to be expressed in bacterial systems whereenzymes needed to remove intervening sequences are absent. Cloned Vregion segments are excised and ligated to L or H chain C regionvectors. Alternatively, the human Cgamma-I region can be modified byintroducing a termination codon thereby generating a gene sequence whichencodes the H chain portion of an Fab molecule. The coding sequenceswith linked V and C regions are then transferred into appropriateexpression vehicles for expression in appropriate hosts, prokaryotic oreukaryotic.

[0144] Two coding DNA sequences are said to be “operably linked” if thelinkage results in a continuously translatable sequence withoutalteration or interruption of the triplet reading frame. A DNA codingsequence is operably linked to a gene expression element if the linkageresults in the proper function of that gene expression element to resultin expression of the coding sequence.

[0145] Expression vehicles include plasmids or other vectors. Preferredamong these are vehicles carrying a functionally complete human C_(H) orC_(L) chain sequence having appropriate restriction sites engineered sothat any V_(H) or V_(L) chain sequence with appropriate cohesive endscan be easily inserted therein. Human C_(H) or C_(L) chainsequence-containing vehicles thus serve as intermediates for theexpression of any desired complete H or L chain in any appropriate host.

[0146] A chimeric antibody, such as a mouse-human or human-human, willtypically be synthesized from genes driven by the chromosomal genepromoters native to the mouse H and L chain V regions used in theconstructs; splicing usually occurs between the splice donor site in themouseJ region and the splice acceptor site preceding the human C regionand also at the splice regions that occur within the human C region;polyadenylation and transcription termination occur at nativechromosomal sites downstream of the human coding regions.

[0147] Non-Limiting Exemplary Chimeric A2 (cA2) Anti-TNF Antibody of thePresent Invention

[0148] Murine MAbs are undesirable for human therapeutic use, due to ashort free circulating serum half-life and the stimulation of a humananti-murine antibody (HAMA) response. A murine-human chimeric anti-humanTNFα MAb was developed in the present invention with high affinity,epitope specificity and the ability to neutralize the cytotoxic effectsof human TNF. Chimeric A2 anti-TNF consists of the antigen bindingvariable region of the high-affinity neutralizing mouse antihuman TNFIgG1 antibody, designated A2, and the constant regions of a human IgG1 ,kappa immunoglobulin. The human IgG1 Fc region is expected to: improveallogeneic antibody effector function; increase the circulating serumhalf-life; and decrease the immunogenicity of the antibody. A similarmurine-human chimeric antibody (chimeric 17-1A) has been shown inclinical studies to have a 6-fold longer in vivo circulation time and tobe significantly less immunogenic than its corresponding murine MAbcounterpart (LoBuglio et al., Proc Natl Acad Sci USA 86: 4220-4224,(1988)).

[0149] The avidity and epitope specificity of the chimeric A2 is derivedfrom the variable region of the murine A2. In a solid phase ELISA,cross-competition for TNF was observed between chimeric and murine A2,indicating an identical epitope specificity of cA2 and murine A2. Thespecificity of cA2 for TNF-α was confirmed by its inability toneutralize the cytotoxic effects of lymphotoxin (TNF-β). Chimeric A2neutralizes the cytotoxic effect of both natural and recombinant humanTNF in a dose dependent manner. From binding assays of cA2 andrecombinant human TNF, the affinity constant of cA2 was calculated to be1.8×10⁹ M¹.

ANTI-TNF Immunoreceptor Peptides

[0150] Immunoreceptor peptides of this invention can bind to TNFα and/orTNFβ. The immunoreceptor comprises covalently attached to at least aportion of the TNF receptor at least one immunoglobulin heavy or lightchain. In certain preferred embodiments, the heavy chain constant regioncomprises at least a portion of CH₁. Specifically, where a light chainis included with an immunoreceptor peptide, the heavy chain must includethe area of CH, responsible for binding a light chain constant region.

[0151] An immunoreceptor peptide of the present invention can preferablycomprise at least one heavy chain constant region and, in certainembodiments, at least one light chain constant region, with a receptormolecule covalently attached to at least one of the immunoglobulinchains. Light chain or heavy chain variable regions are included incertain embodiments. Since the receptor molecule can be linked withinthe interior of an immunoglobulin chain, a single chain can have avariable region and a fusion to a receptor molecule.

[0152] The portion of the TNF receptor linked to the immunoglobulinmolecule is capable of binding TNFα and/or TNFβ. Since the extracellularregion of the TNF receptor binds TNF, the portion attached to theimmunoglobulin molecule of the immunoreceptor consists of at least aportion of the extracellular region of the TNF receptor. In certainpreferred embodiments, the entire extracellular region of p55 isincluded. In other preferred embodiments, the entire extracellularregion of p75 is included. In further preferred embodiments, theextracellular region of p75 is truncated to delete at least a portion ofa region of 0-linked glycosylation and/or a proline-rich region whileleaving intact the intramolecular disulfide bridges. Suchimmunoreceptors comprise at least a portion of a hinge region wherein atleast one heavy chain is covalently linked to a truncated p75extracellular region capable of binding to TNFα or TNFβ or both. Such atruncated molecule includes, for example, sequences 1-178, 1-182 or atleast 5 amino acid portions thereof, such as 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, . . . 50, 60, 70, 80, 90, 100, 110, 120, 130,140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270,280, 290.

[0153] Certain embodiments can also include, for example, the C-terminalhalf of the hinge region to provide a disulfide bridge between heavychains where both CH₂ and CH₃ chains are present and CH₁ is absent.Alternatively, for example, the N-terminal half of the hinge region canbe included to provide a disulfide bridge with a light chain where onlythe CH₁ region is present.

[0154] In certain preferred embodiments of this invention, thenon-immunoglobulin molecule is covalently linked to the N-terminus of atleast one CH₁ region. In other preferred embodiments, thenon-immunoglobulin molecule is covalently linked to an interior sectionof at least one heavy and/or light chain region. Thus, a portion of theTNF receptor can be, for example, at the end of the immunoglobulin chainor in the middle of the chain.

[0155] Where the TNF receptor is attached to the middle of theimmunoglobulin, the immunoglobulin chain can be truncated, for example,to compensate for the presence of foreign amino acids, thus resulting ina fusion molecule of approximately the same length as a naturalimmunoglobulin chain. Alternatively, for example, the immunoglobulinchain can be present substantially in its entirety, thus resulting in achain that is longer than the corresponding natural immunoglobulinchain. Additionally, the immunoglobulin molecule can be truncated toresult in a length intermediate between the size of the entire chainlinked to the receptor molecule and the size of the immunoglobulin chainalone.

[0156] In certain preferred embodiments, the heavy chain is an IgG classheavy chain. In other preferred embodiments, the heavy chain is an IgMclass heavy chain.

[0157] In certain preferred embodiments, the heavy chain furthercomprises at least about 8 amino acids of a J region.

[0158] In certain preferred embodiments, at least a portion of the hingeregion is attached to the CH₁ region. For example, where CH₁ and CH₂ arepresent in the molecule, the entire hinge region is also present toprovide the disulfide bridges between the two heavy chain molecules andbetween the heavy and light chains. Where only CH₁ is present, forexample, the molecule need only contain the portion of the hinge regioncorresponding to the disulfide bridge between the light and heavychains, such as the first 7 amino acids of the hinge.

[0159] It will be understood by one skilled in the art, once armed withthe present disclosure, that the immunoreceptor peptides of theinvention can be, for example, monomeric or dimeric. For example, themolecules can have only one light chain and one heavy chain or two lightchains and two heavy chains.

[0160] At least one of the non-immunoglobulin molecules linked to animmunoglobin molecule comprises at least a portion of p55 or at least aportion of p75. The portion of the receptor that is included encompassesthe TNF binding site.

[0161] In certain preferred embodiments, the non-immunoglobulin moleculecomprises at least 5 amino acid segments of sequences 2-159 of p55. Inother preferred embodiments, the non-immunoglobulin molecule comprisesat least 5 amino acid portions of sequences 1-235 of p75. In furtherpreferred embodiments, the non-immunoglobulin molecule comprises atleast 5 amino acid portions of sequences 1-182 of p75. The above 5 aminoacid portions can be selected from 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160,170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290.

[0162] In certain preferred embodiments, each of the two heavy chainsand each of the two light chains is linked to a portion of the TNFreceptor, thus forming a tetravalent molecule. Such a tetravalentmolecule can have, for example, four p55 receptor molecules; two on thetwo heavy chains and two on the two light chains. Alternatively, atetravalent molecule can have, for example, a p55 receptor moleculeattached to each of the two heavy chains and a p75 receptor moleculeattached to each of the two light chains. A tetravalent molecule canalso have, for example, p55 receptor attached to the light chains andp75 receptor attached to the heavy chains. Additionally, a tetravalentmolecule can have one heavy chain attached to p55, one heavy chainattached to p75, one light chain attached to p75, and one light chainattached to p55. See, for example, the molecules depicted in FIG. 26B.Further, the molecules can have six receptors attached, for example, twowithin the heavy chains and four at the ends of the heavy and lightchains. Other potential multimers and combinations would also be withinthe scope of one skilled in the art, once armed with the presentdisclosure.

[0163] In further preferred embodiments, at least one of the heavychains has a variable region capable of binding to a second targetmolecule. Such molecules include, for example, CD3, so that one half ofa fusion molecule is a monomeric anti-CD3 antibody.

[0164] Additionally, in other embodiments of the present invention, theimmunoreceptor peptides further include an irrelevant variable region onthe light chain and/or heavy chain. Preferably, however, such a regionis absent due to the lowered affinity for TNF which can be present dueto steric hindrance.

[0165] In certain preferred embodiments, the heavy chain is linked to anon-immunoglobulin molecule capable of binding to a second targetmolecule, such as a cytotoxic protein, thus creating a partimmunoreceptor, part immunotoxin that is capable of killing those cellsexpressing TNF. Such cytotoxic proteins, include, but are not limitedto, Ricin-A, Pseudomonas toxin, Diphtheria toxin and TNF. Toxinsconjugated to ligands are known in the art (see, for example, Olsnes, S.et al., Immunol. Today 1989, 10, 291-295). Plant and bacterial toxinstypically kill cells by disruption of the protein synthetic machinery.

[0166] The immunoreceptors of this invention can be conjugated toadditional types of therapeutic moieties including, but not limited to,radionuclides, cytotoxic agents and drugs. Examples of radionuclidesinclude ²¹²Bi, ¹³¹I, ¹⁸⁶Re, and ⁹⁰Y, which list is not intended to beexhaustive. The radionuclides exert their cytotoxic effect by locallyirradiating the cells, leading to various intracellular lesions, as isknown in the art of radiotherapy.

[0167] Cytotoxic drugs which can be conjugated to the immunoreceptorsand subsequently used for in vivo therapy include, but are not limitedto, daunorubicin, doxorubicin, methotrexate, and Mitomycin C. Cytotoxicdrugs interfere with critical cellular processes including DNA, RNA, andprotein synthesis. For a fuller exposition of these classes of drugswhich are known in the art, and their mechanisms of action, see Goodman,A. G., et al., Goodman and Gilman 's The Pharmacological Basis ofTherapeutics, 8th Ed., Macmillan Publishing Col, 1990. Katzung, ed.,Basic and Clinical Pharmacology, Fifth Edition, p 768-769, 808-809, 896,Appleton and Lange, Norwalk, Conn.

[0168] In preferred embodiments, immunoreceptor molecules of theinvention are capable of binding with high affinity to a neutralizingepitope of human TNFα or TNFβ in vivo. Preferably, the binding affinityis at least about 1.6×10¹⁰ ¹⁰ M-1. Additionally, in preferredembodiments, immunoreceptor molecules of the invention are capable ofneutralizing TNF at an efficiency of about a concentration of less than130 pM to neutralize 39.2 pM human TNFα. See, for example, Table 1.TABLE 1 Summary of affinities of different fusion proteins for TNFα.Molar ratio fp: TNFα at Protein IC₅₀* IC₅₀ K_(D) (pM) p55-sf2 70 1.8 57p55-df2 55 1.4 60 p55-sf3 100 2.6 48 p55-nf 36,000 900 n.d. p75-sf2 1303.3 33 p75P-sf2 70 1.8 29 p75P-sf3 130 3.3 15

[0169] Once armed with the present disclosure, one skilled in the artwould be able to create fragments of the imimunoreceptor peptides of theinvention. Such fragments are intended to be within the scope of thisinvention. For example, once the molecules are isolated, they can becleaved with protease to generate fragments that remain capable ofbinding TNF.

[0170] Once armed with the present disclosure, one skilled in the artwould also be able to create derivatives of the immunoreceptor peptidesof the invention. Such derivatives are intended to be within the scopeof this invention. For example, amin o acids in the immunoreceptor thatconstitute a protease recognition site can be modified to avoid proteasecleavage and thus confer greater stability, such as KEX2 sites.

[0171] One skilled in the art, once armed with the present disclosure,would be able to synthesize the molecules of the invention. Theimmunoreceptor peptides can be constructed, for example, byvector-mediated synthesis, as described in Example XXVI. In general, twoexpression vectors are preferably used; one for the heavy chain, one forthe light chain. A vector for expression of an immunoglobulin preferablyconsists of a promoter linked to the signal sequence, followed by theconstant region. The vector additionally preferably contains a geneproviding for the selection of transfected cells expressing theconstruct. In certain preferred embodiments, sequences derived from theJ region are also included.

[0172] The immunoglobulin gene can be from any vertebrate source, suchas murine, but preferably, it encodes an immunoglobulin having asubstantial number of sequences that are of the same origin as theeventual recipient of the immunoreceptor peptide. For example, if ahuman is treated with a molecule of the invention, preferably theimmunoglobulin is of human origin.

[0173] TNF receptor constructs for linking to the heavy chain can besynthesized, for example, using DNA encoding amino acids present in thecellular domain of the receptor. Putative receptor binding loci of hTNFhave been presented by Eck and Sprange,J. Biol. Chem. 264(29),17595-17605 (1989), who identified the receptor binding loci of TNFα asconsisting of amino acids 11-13, 37-42, 49-57 and 155-157. PCTapplication WO91/02078 (priority date of Aug. 7, 1989) discloses TNFligands which can bind to monoclonal antibodies having the followingepitopes of at least one of 1-20, 56-77, and 108-127; at least two of1-20, 56-77, 108-127 and 138-149; all of 1-18, 58-65, 115-125 and138-149; all of 1-18, and 108-128; all of 56-79, 110-127 and 135- or136-155; all of 1-30, 117-128 and 141-153; all of 1-26, 117-128 and141-153; all of 22-40, 49-96 or -97, 110-127 and 136-153; all of 12-22,36-45, 96-105 and 132-157; all of both of 1-20 and 76-90; all of 22-40,69-97, 105-128 and 135-155; all of 22-31 and 146-157; all of 22-40 and49-98; at least one of 22-40, 9-98 and 69-97, both of 22-40 and 70-87.Thus, one skilled in the art, once armed with the present disclosure,would be able to create TNF receptor fusion proteins using portions ofthe receptor that are known to bind TNF.

[0174] Advantages of using an immunoglobulin fusion protein(immunoreceptor peptide) of the present invention include one or more of(1) possible increased avidity for multivalent ligands due to theresulting bivalency of dimeric fusion proteins, (2) longer serumhalf-life, (3) the ability to activate effector cells via the Fc domain,(4) ease of purification (for example, by protein A chromatography), (5)affinity for TNFα and TNFβ and (6) the ability to block TNFα or TNFβcytotoxicity.

[0175] TNF receptor/IgG fusion proteins have shown greater affinity forTNFα in vitro than their monovalent, non-fusion counterparts. Thesetypes of fusion proteins, which also bind murine TNF with high affinity,have also been shown to protect mice from lipopolysaccharide-inducedendotoxemia. Lesslauer et al., Eur. J. ImmunoL 21, 2883-2886 (1991); andAshkenazi et al., Proc. Natl. Acad. Sci. USA 88:10535-10539 (1991).Unlike the molecules of the present invention, the TNF receptor/IgGfusion proteins reported to date have had the receptor sequence fuseddirectly to the hinge domain of IgGs such that the first constant domain(CHI) of the heavy chain was omitted. Lesslauer et al., Eur. J. Immunol.1:2883-2886; Ashkenzi et al., Proc. Natl. Acad. Sci. USA 88:10535-10539(1991); and Peppel et al.,J. Exp. Med. 174:1483-1489 (1991).

[0176] While this generally permits secretion of the fusion protein inthe absence of an Ig light chain, a major embodiment of the presentinvention provides for the inclusion of the CH, domain, which can conferadvantages such as (1) increased distance and/or flexibility between tworeceptor molecules resulting in greater affinity for TNF, (2) theability to create a heavy chain fusion protein and a light chain fusionprotein that would assemble with each other and dimerize to form atetravalent (double fusion) receptor molecule, and (3) a tetravalentfusion protein can have increased affinity and/or neutralizingcapability for TNF compared to a bivalent (single fusion) molecule.

[0177] Unlike other TNF receptor/IgG fusion proteins that have beenreported, the fusion proteins of a major embodiment of the presentinvention include the first constant domain (CH₁) of the heavy chain.The CH, domain is largely responsible for interactions with lightchains. The light chain, in turn, provides a vehicle for attaching asecond set of TNF receptor molecules to the immunoreceptor peptide.

[0178] It was discovered using the molecules of the present inventionthat the p55/light chain fusion proteins and p55/heavy chain fusionproteins would assemble with each other and dimerize to form anantibody-like molecule that is tetravalent with respect to p55. Theresulting tetravalent p55 molecules can confer more protection against,and have greater affinity for, TNFα or TNFβ than the bivalent p55molecules. Despite the presumed close proximity of the two light chainp55 domains to the heavy chain p55 domains, they do not appearsterically hinder or reduce the affinity for TNF.

[0179] Inclusion of the CH₁ domain also meant that secretion of thefusion protein was likely to be inefficient in the absence of lightchain. This has been shown to be due to a ubiquitous immunoglobulinbinding protein (BiP) that binds to the CH, domain of heavy chains thatare not assembled with a light chain and sequesters them in theendoplasmic reticulum. Karlsson et al.,J. Immunol. Methods 145:229-240(1991).

[0180] In initial experiments, an irrelevant light chain wasco-transfected with the p55-heavy chain construct and subsequentanalyses showed that the two chains did assemble and that the resultingfusion protein protected WEHI cells from TNFα. However, it wasconsidered likely that the variable region of the irrelevant light chainwould sterically hinder interactions between the p55 subunits and TNFα.For this reason, a mouse-human chimeric antibody light chain gene wasengineered by (1) deleting the variable region coding sequence, and (2)replacing the murine J coding sequence with human J coding sequence. Useof this truncated light chain, which was shown to assemble and disulfidebond with heavy chains, increased the efficiency of TNF inhibition byapproximately 30-fold compared to use of a complete irrelevant lightchain.

[0181] Comparison of the abilities of p75-sf2 and p75P-sf2 to inhibitTNF cytotoxicity indicated that the C-terminal 53 amino acids of theextracellular domain of p75, which defines a region that is rich inproline residues and contains the only sites of 0-linked glycosylation,are not necessary for high-affinity binding to TNFα or TNFβ. In fact,the p75P-sf2 construct repeatedly showed higher affmity binding to TNFβthan p75-sf2. Surprisingly, there was no difference observed between thetwo constructs in their affinity for TNFα.

[0182] It is possible that a cell-surface version of p75-P would alsobind TNFβ with higher affinity than the complete p75 extracellulardomain. A similar region is found adjacent to the transmembrane domainin the low affinity nerve growth factor receptor whose extracellulardomain shows the same degree of similarity to p75 as p55 does. Mallerret al., Immunol. Today 12:220-223 (1991).

[0183] Two groups have reported that in cell cytotoxicity assays, theirp55 fusion protein could be present at a 3-fold (Lesslauer et al., Eur.J. Immunol. 21:2883-2886 (1991)) or 6-8 fold (Ashkenazi et al., Proc.Natl. Acad. Sci. USA 88:10535-10539 (1991)) lower concentration thantheir monovalent p55 and still get the same degree of protection, whileanother group (Peppel et al.,J. Exp. Med. 174:1483-1489 (1991)) showedthat their p55 fusion protein could be present at a 1000-fold lowerconcentration than monomeric p55. Thus, the prior art has shownunpredictability in the great variability in the efficiency of differentfusion proteins.

[0184] The molecules of the present invention have demonstrated the samedegree of protection against TNF in a 5000-fold lower molarconcentration than monomeric p55. (See Table 1.) It is believed that thepresence of the CH₁ chain in the molecules of a major embodiment of thepresent invention can confer greater flexibility to the molecule andavoid steric hindrance with the binding of the TNF receptor.

Recombinant Expression of Anti-TNF Antibodies and Anti-TNF Peptides

[0185] A nucleic acid sequence encoding at least one anti-TNF peptide orAb fragment of the present invention may be recombined with vector DNAin accordance with conventional techniques, including blunt-ended orstaggered-ended termini for ligation, restriction enzyme digestion toprovide appropriate termini, filling in of cohesive ends as appropriate,alkaline phosphatase treatment to avoid undesirable joining, andligation with appropriate ligases. Techniques for such manipulations aredisclosed, e.g., by Ausubel, infra, Sambrook, infra, entirelyincorporated herein by reference, and are well known in the art.

[0186] A nucleic acid molecule, such as DNA, is said to be “capable ofexpressing” a polypeptide if it contains nucleotide sequences whichcontain transcriptional and translational regulatory information andsuch sequences are “operably linked” to nucleotide sequences whichencode the polypeptide. An operable linkage is a linkage in which theregulatory DNA sequences and the DNA sequence sought to be expressed areconnected in such a way as to permit gene expression as anti-TNFpeptides or Ab fragments in recoverable amounts. The precise nature ofthe regulatory regions needed for gene expression may vary from organismto organism, as is well known in the analogous art. See, e.g., Sambrook,supra and Ausubel supra.

[0187] The present invention accordingly encompasses the expression ofan anti-TNF peptide or Ab fragment, in either prokaryotic or eukaryoticcells, although eukaryotic expression is preferred.

[0188] Preferred hosts are bacterial or eukaryotic hosts includingbacteria, yeast, insects, fungi, bird and mammalian cells either invivo, or in situ, or host cells of mammalian, insect, bird or yeastorigin. It is preferred that the mammalian cell or tissue is of human,primate, hamster, rabbit, rodent, cow, pig, sheep, horse, goat, dog orcat origin, but any other mammalian cell may be used.

[0189] Further, by use of, for example, the yeast ubiquitin hydrolasesystem, in vivo synthesis of ubiquitin-transmembrane polypeptide fusionproteins may be accomplished. The fusion proteins so produced may beprocessed in vivo or purified and processed in vitro, allowing synthesisof an anti-TNF peptide or Ab fragment of the present invention with aspecified amino terminus sequence. Moreover, problems associated withretention of initiation codon-derived methionine residues in directyeast (or bacterial) expression maybe avoided. Sabin et al.,Bio/Technol. 7(7): 705-709 (1989); Miller et al., Bio/Technol. 7(7):698-704 (1989).

[0190] Any of a series of yeast gene expression systems incorporatingpromoter and termination elements from the actively expressed genescoding for glycolytic enzymes produced in large quantities when yeastare grown in mediums rich in glucose can be utilized to obtain anti-TNFpeptides or Ab fragments of the present invention. Known glycolyticgenes can also provide very efficient transcriptional control signals.For example, the promoter and terminator signals of the phosphoglyceratekinase gene can be utilized.

[0191] Production of anti-TNF peptides or Ab fragments or functionalderivatives thereof in insects can be achieved, for example, byinfecting the insect host with a baculovirus engineered to express atransmembrane polypeptide by methods known to those of skill. SeeAusubel et al., eds. Current Protocols in Molecular Biology WileyInterscience, §§16.8-16.11 (1987, 1993).

[0192] In a preferred embodiment, the introduced nucleotide sequencewill be incorporated into a plasmid or viral vector capable ofautonomous replication in the recipient host. Any of a wide variety ofvectors may be employed for this purpose. See, e.g., Ausubel et al.,infra, §§ 1.5, 1.10, 7.1, 7.3, 8.1, 9.6, 9.7, 13.4, 16.2, 16.6, and16.8-16.11. Factors of importance in selecting a particular plasmid orviral vector include: the ease with which recipient cells that containthe vector may be recognized and selected from those recipient cellswhich do not contain the vector; the number of copies of the vectorwhich are desired in a particular host; and whether it is desirable tobe able to “shuttle” the vector between host cells of different species.

[0193] Preferred prokaryotic vectors known in the art include plasmidssuch as those capable of replication in E. coli (such as, for example,pBR322, Co1E1, pSC101, pACYC 184, πVX). Such plasmids are, for example,disclosed by Maniatis, T., et al. (Molecular Cloning, A LaboratoryManual, Second Edition, Cold Spring Harbor Press, Cold Spring Harbor,N.Y. (1989); Ausubel, infra. Bacillus plasmids include pC194, pC221,pT127, etc. Such plasmids are disclosed by Gryczan, T. (In: TheMolecular Biology of the Bacilli, Academic Press, NY (1982), pp.307-329). Suitable Streptomyces plasmids include pIJ101 (Kendall, K. J.,et al.,J. Bacteriol. 169:4177-4183 (1987)), and streptomycesbacteriophages such as φC31 (Chater, K. F., et al., In: SixthInternational Symposium on Actinomycetales Biology, Akademiai Kaido,Budapest, Hungary (1986), pp. 45-54). Pseudomonas plasmids are reviewedby John, J. F., et al. (Rev. Infect. Dis. 8:693-704 (1986)), and Izaki,K. (Jpn.J. Bacteriol. 33:729-742 (1978); and Ausubel et al., supra).

[0194] Alternatively, gene expression elements useful for the expressionof cDNA encoding anti-TNF antibodies or peptides include, but are notlimited to (a) viral transcription promoters and their enhancerelements, such as the SV40 early promoter (Okayama, et al., Mol. Cell.Biol. 3:280 (1983)), Rous sarcoma virus LTR (Gorman, et al., Proc. Natl.Acad. Sci., USA 79:6777 (1982)), and Moloney murine leukemia virus LTR(Grosschedl, et al., Cell 41:885 (1985)); (b) splice regions andpolyadenylation sites such as those derived from the SV40 late region(Okayarea et al., infra); and (c) polyadenylation sites such as in SV40(Okayama et al., infra).

[0195] Immunoglobulin cDNA genes can be expressed as described by Liu etal., infra, and Weidle et al., Gene 51:21 (1987), using as expressionelements the SV40 early promoter and its enhancer, the mouseimmunoglobulin H chain promoter enhancers, SV40 late region mRNAsplicing, rabbit S-globin intervening sequence, immunoglobulin andrabbit S-globin polyadenylation sites, and SV40 polyadenylationelements.

[0196] For immunoglobulin genes comprised of part cDNA, part genomic DNA(Whittle et al., Protein Engineering 1:499 (1987)), the transcriptionalpromoter can be human cytomegalovirus, the promoter enhancers can becytomegalovirus and mouse/human immunoglobulin, and mRNA splicing andpolyadenylation regions can be the native chromosomal immunoglobulinsequences.

[0197] In one embodiment, for expression of cDNA genes in rodent cells,the transcriptional promoter is a viral LTR sequence, thetranscriptional promoter enhancers are either or both the mouseimmunoglobulin heavy chain enhancer and the viral LTR enhancer, thesplice region contains an intron of greater than 31 bp, and thepolyadenylation and transcription termination regions are derived fromthe native chromosomal sequence corresponding to the immunoglobulinchain being synthesized. In other embodiments, cDNA sequences encodingother proteins are combined with the above-recited expression elementsto achieve expression of the proteins in mammalian cells.

[0198] Each fused gene is assembled in, or inserted into, an expressionvector. Recipient cells capable of expressing the chimericimmunoglobulin chain gene product are then transfected singly with ananti-TNF peptide or chimeric H or chimeric L chain-encoding gene, or areco-transfected with a chimeric H and a chimeric L chain gene. Thetransfected recipient cells are cultured under conditions that permitexpression of the incorporated genes and the expressed immunoglobulinchains or intact antibodies or fragments are recovered from the culture.

[0199] In one embodiment, the fused genes encoding the anti-TNF peptideor chimeric H and L chains, or portions thereof, are assembled inseparate expression vectors that are then used to co-transfect arecipient cell.

[0200] Each vector can contain two selectable genes, a first selectablegene designed for selection in a bacterial system and a secondselectable gene designed for selection in a eukaryotic system, whereineach vector has a different pair of genes. This strategy results invectors which first direct the production, and permit amplification, ofthe fused genes in a bacterial system. The genes so produced andamplified in a bacterial host are subsequently used to co-transfect aeukaryotic cell, and allow selection of a co-transfected cell carryingthe desired transfected genes.

[0201] Examples of selectable genes for use in a bacterial system arethe gene that confers resistance to ampicillin and the gene that confersresistance to chloramphenicol. Preferred selectable genes for use ineukaryotic transfectants include the xanthine guanine phosphoribosyltransferase gene (designated gpt) and the phosphotransferase gens fromTn5 (designated neo).

[0202] Selection of cells expressing gpt is based on the fact that theenzyme encoded by this gene utilizes xanthine as a substrate for purinenucleotide synthesis, whereas the analogous endogenous enzyme cannot. Ina medium containing (1) mycophenolic acid, which blocks the conversionof inosine monophosphate to xanthine monophosphate, and (2) xanthine,only cells expressing the gpt gene can survive. The product of the neoblocks the inhibition of protein synthesis by the antibiotic G418 andother antibiotics of the neomycin class.

[0203] The two selection procedures can be used simultaneously orsequentially to select for the expression of immunoglobulin chain genesintroduced on two different DNA vectors into a eukaryotic cell. It isnot necessary to include different selectable markers for eukaryoticcells; an H and an L chain vector, each containing the same selectablemarker can be co-transfected. After selection of the appropriatelyresistant cells, the majority of the clones will contain integratedcopies of both H and L chain vectors and/or anti-TNF peptides.

[0204] Alternatively, the fused genes encoding the chimeric H and Lchains can be assembled on the same expression vector.

[0205] For transfection of the expression vectors and production of thechimeric antibody, the preferred recipient cell line is a myeloma cell.Myeloma cells can synthesize, assemble and secrete immunoglobulinsencoded by transfected immunoglobulin genes and possess the mechanismfor glycosylation of the immunoglobulin. A particularly preferredrecipient cell is the recombinant Ig-producing myeloma cell SP2/0 (ATCC#CRL 8287). SP2/0 cells produce only immunoglobulin encoded by thetransfected genes. Myeloma cells can be grown in culture or in theperitoneal cavity of a mouse, where secreted immunoglobulin can beobtained from ascites fluid. Other suitable recipient cells includelymphoid cells such as B lymphocytes of human or non-human origin,hybridoma cells of human or non-human origin, or interspeciesheterohybridoma cells.

[0206] The expression vector carrying a chimeric antibody construct oranti-TNF peptide of the present invention can be introduced into anappropriate host cell by any of a variety of suitable means, includingsuch biochemical means as transformation, transfection, conjugation,protoplast fusion, calcium phosphate-precipitation, and application withpolycations such as diethylaminoethyl (DEAE) dextran, and suchmechanical means as electroporation, direct microinjection, andmicroprojectile bombardment (Johnston et al., Science 240:1538 (1988)).A preferred way of introducing DNA into lymphoid cells is byelectroporation (Potter et al., Proc. Natl. Acad. Sci. USA 81:7161(1984); Yoshikawa, et al., Jpn. J. Cancer Res. 77:1122-1133). In thisprocedure, recipient cells are subjected to an electric pulse in thepresence of the DNA to be incorporated. Typically, after transfection,cells are allowed to recover in complete medium for about 24 hours, andare then seeded in 96-well culture plates in the presence of theselective medium. G418 selection is performed using about 0.4 to 0.8mg/ml G418. Mycophenolic acid selection utilizes about 6 μg/ml plusabout 0.25 mg/ml xanthine. The electroporation technique is expected toyield transfection frequencies of about 10⁵ to about 10 for Sp2/0 cells.In the protoplast fusion method, lysozyme is used to strip cell wallsfrom catarrhal harboring the recombinant plasmid containing the chimericantibody gene. The resulting spheroplasts are fused with myeloma cellswith polyethylene glycol.

[0207] The immunoglobulin genes of the present invention can also beexpressed in nonlymphoid mammalian cells or in other eukaryotic cells,such as yeast, or in prokaryotic cells, in particular bacteria.

[0208] Yeast provides substantial advantages over bacteria for theproduction of immunoglobulin H and L chains. Yeasts carry outpost-translational peptide modifications including glycosylation. Anumber of recombinant DNA strategies now exist which utilize strongpromoter sequences and high copy number plasmids which can be used forproduction of the desired proteins in yeast. Yeast recognizes leadersequences of cloned mammalian gene products and secretes peptidesbearing leader sequences (i.e., pre-peptides) (Hitzman, et al., 11thInternational Conference on Yeast, Genetics and Molecular Biology,Montpelier, France, Sep. 13-17, 1982).

[0209] Yeast gene expression systems can be routinely evaluated for thelevels of production, secretion and the stability of anti-TNF peptides,antibody and assembled murine and chimeric antibodies, fragments andregions thereof. Any of a series of yeast gene expression systemsincorporating promoter and termination elements from the activelyexpressed genes coding for glycolytic enzymes produced in largequantities when yeasts are grown in media rich in glucose can beutilized. Known glycolytic genes can also provide very efficienttranscription control signals. For example, the promoter and terminatorsignals of the phosphoglycerate kinase (PGK) gene can be utilized. Anumber of approaches can be taken for evaluating optimal expressionplasmids for the expression of cloned immunoglobulin cDNAs in yeast (seeGlover, ed., DNA Cloning, Vol. II, pp45-66, ML Press, 1985).

[0210] Bacterial strains can also be utilized as hosts for theproduction of antibody molecules or peptides described by thisinvention, E. coli K12 strains such as E. coli W3110 (ATCC 27325), andother enterobacteria such as Salmonella typhimurium or Serratiamarcescens, and various Pseudomonas species can be used.

[0211] Plasmid vectors containing replicon and control sequences whichare derived from species compatible with a host cell are used inconnection with these bacterial hosts. The vector carries a replicationsite, as well as specific genes which are capable of providingphenotypic selection in transformed cells. A number of approaches can betaken for evaluating the expression plasmids for the production ofmurine and chimeric antibodies, fragments and regions or antibody chainsencoded by the cloned immunoglobulin cDNAs in bacteria (see Glover, ed.,DNA Cloning, Vol I, IRI Press, 1985, Ausubel, infra, Sambrook, infra,Colligan, infra).

[0212] Preferred hosts are mammalian cells, grown in vitro or in vivo.Mammalian cells provide post-translational modifications toimmunoglobulin protein molecules including leader peptide removal,folding and assembly of H and L chains, glycosylation of the antibodymolecules, and secretion of finctional antibody protein.

[0213] Mammalian cells which can be useful as hosts for the productionof antibody proteins, in addition to the cells of lymphoid origindescribed above, include cells of fibroblast origin, such as Vero (ATCCCRL 81) or CHO-KL (ATCC CRL 61).

[0214] Many vector systems are available for the expression of clonedanti-TNF peptides H and L chain genes in mammalian cells (see Glover,ed., DNA Cloning, Vol. II, ppl43-238, IkL Press, 1985). Differentapproaches can be followed to obtain complete H₂L₂ antibodies. Asdiscussed above, it is possible to co-express H and L chains in the samecells to achieve intracellular association and linkage of H and L chainsinto complete tetrameric H₂L₂ antibodies and/or anti-TNF peptides. Theco-expression can occur by using either the same or different plasmidsin the same host. Genes for both H and L chains and/or anti-TNF peptidescan be placed into the same plasmid, which is then transfected intocells, thereby selecting directly for cells that express both chains.Alternatively, cells can be transfected first with a plasmid encodingone chain, for example the L chain, followed by transfection of theresulting cell line with an H chain plasmid containing a secondselectable marker. Cell lines producing anti-TNF peptides and/or H₂L₂molecules via either route could be transfected with plasmids encodingadditional copies of peptides, H, L, or H plus L chains in conjunctionwith additional selectable markers to generate cell lines with enhancedproperties, such as higher production of assembled H₂L₂ antibodymolecules or enhanced stability of the transfected cell lines.

Anti-idiotype Abs

[0215] In addition to monoclonal or chimeric anti-TNF antibodies, thepresent invention is also directed to an anti-idiotypic (anti-Id)antibody specific for the anti-TNF antibody of the invention. An anti-Idantibody is an antibody which recognizes unique determinants generallyassociated with the antigen-binding region of another antibody. Theantibody specific for TNF is termed the idiotypic or Id antibody. Theanti-Id can be prepared by immunizing an animal of the same species andgenetic type (e.g. mouse strain) as the source of the Id antibody withthe Id antibody or the antigen-binding region thereof. The immunizedanimal will recognize and respond to the idiotypic determinants of theimmunizing antibody and produce an anti-Id antibody. The anti-Idantibody can also be used as an “immunogen” to induce an immune responsein yet another animal, producing a so-called anti-anti-Id antibody. Theanti-anti-Id can be epitopically identical to the original antibodywhich induced the anti-Id. Thus, by using antibodies to the idiotypicdeterminants of a mAb, it is possible to identify other clonesexpressing antibodies of identical specificity.

[0216] Accordingly, mAbs generated against TNF according to the presentinvention can be used to induce anti-Id antibodies in suitable animals,such as BALB/c mice. Spleen cells from such immunized mice can be usedto produce anti-Id hybridomas secreting anti-Id mAbs. Further, theanti-Id mAbs can be coupled to a carrier such as keyhole limpethemocyanin (KLH) and used to immunize additional BALB/c mice. Sera fromthese mice will contain anti-anti-Id antibodies that have the bindingproperties of the original mAb specific for a TNF epitope.

[0217] Screening Methods for determining tissue necrosis factorneutralizing and/or inhibiting activity are also provided in the presentinvention. In the context of the present invention, TNF neutralizingactivity or TNF inhibiting activity refers to the ability of a TNFneutralizing compound to block at least one biological activity of TNF,such as preventing TNF from binding to a TNF receptor, blockingproduction of TNF by intracellular processing, such as transcription,translation or post-translational modification, expression on the cellsurface, secretion or assembly of the bioactive trimer of TNF.Additionally, TNF neutralizing compounds can act by inducing regulationof metabolic pathways such as those involving the up or down regulationof TNF production. Alternatively TNF neutralizing compounds can modulatecellular sensitivity to TNF by decreasing such sensitivity. TNFneutralizing compounds can be selected from the group consisting ofantibodies, or fragments or portions thereof, peptides, peptido mimeticcompounds or organo mimetic compounds that neutralizes TNF activity invitro, in situ or in vivo is considered a TNF neutralizing compound ifused according to the present invention. Screening methods which can beused to determine TNF neutralizing activity of a TNF neutralizingcompound can include in vitro or in vivo assays. Such in vitro assayscan include a TNF cytotoxicity assay, such as a radioimmuno assay, whichdetermines a decrease in cell death by contact with TNP, such aschimpanzee or human TNF in isolated or recombinant form, wherein theconcurrent presence of a TNF neutralizing compound reduces the degree orrate of cell death. The cell death can be determined using ID50 valueswhich represent the concentration of a TNF neutralizing compound whichdecreases the cell death rate by 50%. For example, MAb's A2 and cA2 arefound to have ID50 about 17 mg/ml+/−3 mg/ml, such as 14-20 mg/ml, or anyrange or value therein. Such a TNF cytotoxicity assay is presented inExample II.

[0218] Alternatively or additionally, another in vitro assay which canbe used to determine neutralizing activity of a TNF neutralizingcompound is an assay which measures the neutralization of TNF inducedprocoagulant activity, such as presented in Example XI.

[0219] Alternatively or additionally, TNF neutralizing activity of a TNFneutralizing compound can be measured by an assay for the neutralizationof TNF induced IL-6 secretion, such as using cultured human umbilicalvein endothelial cells (HUVEC), for example. Also presented in ExampleXI.

[0220] Alternatively or additionally, in vivo testing of TNFneutralizing activity of TNF neutralizing compounds can be tested usingsurvival of a mouse given lethal doses of Rh TNF with controlled andvaried concentrations of a TNF neutralizing compound, such as TNFantibodies. Preferably galactosamine sensitive mice are used. Forexample, using a chimeric human anti-TNF antibody as a TNF neutralizingcompound, a concentration of 0.4 milligrams per kilogram TNF antibodyresulted in a 70-100% increase in survival and a 2.0 mg/kg dose of TNFantibody resulted in a 90-100% increase in survival rate using such anassay, for example, as presented in Example XVI.

[0221] Additionally, after TNF neutralizing compounds are tested forsafety in animal models such as chimpanzees, for example as presented inExample XVII, TNF neutralizing compounds can be used to treat variousTNF related pathologies, as described herein, and as presented inExamples XVII-XXV.

[0222] Accordingly, any suitable TNF neutralizing compound can be usedin methods according to the present invention. Examples of such TNFneutralizing compound can be selected from the group consisting ofantibodies or portions thereof specific to neutralizing epitopes of TNF,p55 receptors, p75 receptors, or complexes thereof, portions of TNFreceptors which bind TNF, peptides which bind TNF, any peptido mimeticdrugs which bind TNF and any organo mimetic drugs that block TNF.

[0223] Such TNF neutralizing compounds can be determined by routineexperimentation based on the teachings and guidance presented herein, bythose skilled in the relevant arts.

Structural Analogs of Anti-TNF Antibodies and Anti-TNF Peptides

[0224] Structural analogs of anti-TNF Abs and peptides of the presentinvention are provided by known method steps based on the teaching andguidance presented herein.

[0225] Knowledge of the three-dimensional structures of proteins iscrucial in understanding how they function. The three-dimensionalstructures of more than 400 proteins are currently available in proteinstructure databases (in contrast to around 15,000 known proteinsequences in sequence databases). Analysis of these structures showsthat they fall into recognizable classes of motifs. It is thus possibleto model a three-dimensional structure of a protein based on theprotein's homology to a related protein of known structure. Manyexamples are known where two proteins that have relatively low sequencehomology, can have very similar three dimensional structures or motifs.

[0226] In recent years it has become possible to determine the threedimensional structures of proteins of up to about 15 kDa by nuclearmagnetic resonance (NMR). The technique only requires a concentratedsolution of pure protein. No crystals or isomorphous derivatives areneeded. The structures of a number of proteins have been determined bythis method. The details of NMR structure determination are well-knownin the art (See, e.g., Wuthrich, NMR of Proteins and Nucleic Acids,Wiley, New York, 1986; Wuthrich, K. Science 243:45-50 (1989); Clore etal., Crit. Rev. Bioch. Molec. Biol. 24:479-564 (1989); Cooke et al.,Bioassays 8: 52-56 (1988), which references are hereby incorporatedherein by reference).

[0227] In applying this approach, a variety of ¹H NMR 2D data sets arecollected for anti-TNF Abs and/or anti-TNF peptides of the presentinvention. These are of two main types. One type, COSY (CorrelatedSpectroscopy) identifies proton resonances that are linked by chemicalbonds. These spectra provide information on protons that are linked bythree or less covalent bonds. NOESY (nuclear Overhauser enhancementspectroscopy) identifies protons which are close in space (less than 0.5nm). Following assignment of the complete spin system, the secondarystructure is defined by NOESY. Cross peaks (nuclear Overhauser effectsor NOE's) are found between residues that are adjacent in the primarysequence of the peptide and can be seen for protons less than 0.5nmapart. The data gathered from sequential NOE's combined with amideproton coupling constants and NOE's from non-adjacent amino acids thatare adjacent to the secondary structure, are used to characterize thesecondary structure of the polypeptides. Aside from predicting secondarystructure, NOE's indicate the distance that protons are in space in boththe primary amino acid sequence and the secondary structures. Tertiarystructure predictions are determined, after all the data are considered,by a “best fit” extrapolation.

[0228] Types of amino acid are first identified using through-bondconnectivities. The second step is to assign specific amino acids usingthrough-space connectivities to neighboring residues, together with theknown amino acid sequence. Structural information is then tabulated andis of three main kinds: The NOE identifies pairs of protons which areclose in space, coupling constants give information on dihedral anglesand slowly exchanging amide protons give information on the position ofhydrogen bonds. The restraints are used to compute the structure using adistance geometry type of calculation followed by refinement usingrestrained molecular dynamics. The output of these computer programs isa family of structures which are compatible with the experimental data(i.e. the set of pairwise <0.5 nm distance restraints). The better thatthe structure is defined by the data, the better the family ofstructures can be superimposed, (i.e., the better the resolution of thestructure). In the better defined structures using NMR, the position ofmuch of the backbone (i.e. the amide, Ca and carbonyl atoms) and theside chains of those amino acids that lie buried in the core of themolecule can be defined as clearly as in structures obtained bycrystallography. The side chains of amino acid residues exposed on thesurface are frequently less well defined, however. This probablyreflects the fact that these surface residues are more mobile and canhave no fixed position. (In a crystal structure this might be seen asdiffuse electron density).

[0229] Thus, according to the present invention, use of NMRspectroscopic data is combined with computer modeling to arrive atstructural analogs of at least portions of anti-TNF Abs and peptidesbased on a structural understanding of the topography. Using thisinformation, one of ordinary skill in the art will know how to achievestructural analogs of anti-TNF Abs and/or peptides, such as byrationally-based amino acid substitutions allowing the production ofpeptides in which the TNF binding affinity is modulated in accordancewith the requirements of the expected therapeutic or diagnostic use ofthe molecule, preferably, the achievement of greater specificity for TNFbinding.

[0230] Alternatively, compounds having the structural and chemicalfeatures suitable as anti-TNF therapeutics and diagnostics providestructural analogs with selective TNF affinity. Molecular modelingstudies of TNF binding compounds, such as TNF receptors, anti-TNFantibodies, or other TNF binding molecules, using a program such asMACROMODEL®, INSIGHT@, and DISCOVER®provide such spatial requirementsand orientation of the anti-TNF Abs and/or peptides according to thepresent invention. Such structural analogs of the present invention thusprovide selective qualitative and quantitative anti-TNF activity invitro, in situ and/or in vivo.

Therapeutic Methods for Treating TNF-Related Pathologies

[0231] The anti-TNF peptides, antibodies, fragments and/or derivativesof the present invention are useful for treating a subject having apathology or condition associated with abnormal levels of a substancereactive with an anti-TNF antibody, in particular TNF, such as TNFα orTNFβ, in excess of levels present in a normal healthy subject, wheresuch excess levels occur in a systemic, localized or particular tissuetype or location in the body. Such tissue types can include, but are notlimited to, blood, lymph, CNS, liver, kidney, spleen, heart muscle orblood vessels, brain or spinal cord white matter or grey matter,cartilage, ligaments, tendons, lung, pancreas, ovary, testes, prostate.Increased TNF concentrations relative to normal levels can also belocalized to specific regions or cells in the body, such as joints,nerve blood vessel junctions, bones, specific tendons or ligaments, orsites of infection, such as bacterial or viral infections.

[0232] TNF related pathologies include, but are not limited to, thefollowing:

[0233] (A) acute and chronic immune and autoimmune pathologies, such assystemic lupus erythematosus (SLE), rheumatoid arthritis, thyroidosis,graft versus host disease, scleroderna, diabetes mellitus, Graves'disease, Beschet's disease, and the like;

[0234] (B) infections, including, but not limited to, sepsis syndrome,cachexia, circulatory collapse and shock resulting from acute or chronicbacterial infection, acute and chronic parasitic and/or infectiousdiseases, bacterial, viral or fungal, such as a HIV, AIDS (includingsymptoms of cachexia, autoimmune disorders, AIDS dementia complex andinfections);

[0235] (C) inflammatory diseases, such as chronic inflammatorypathologies and vascular inflammatory pathologies, including chronicinflammatory pathologies such as sarcoidosis, chronic inflammatory boweldisease, ulcerative colitis, and Crohn's pathology and vascularinflammatory pathologies, such as, but not limited to, disseminatedintravascular coagulation, atherosclerosis, and Kawasaki's pathology:

[0236] (D) neurodegenerative diseases, including, but are not limitedto, demyelinating diseases, such as multiple sclerosis and acutetransverse myelitis; extrapyramidal and cerebellar disorders such aslesions of the corticospinal system; disorders of the basal ganglia orcerebellar disorders; hyperkinetic movement disorders such asHuntington's Chorea and senile chorea; drug-induced movement disorders,such as those induced by drugs which block CNS dopamine receptors;hypokinetic movement disorders, such as Parkinson's disease; Progressivesupranucleo palsy; Cerebellar and Spinocerebellar Disorders, such asastructural lesions of the cerebellum; spinocerebellar degenerations(spinal ataxia, Friedreich's ataxia, cerebellar cortical degenerations,multiple systems degenerations (Mencel, Dejerine-Thomas, Shi-Drager, andMachadoJoseph)); and systemic disorders (Refsum's disease, abetalipoprotemia, ataxia, telangiectasia, and mitochondrial multi-systemdisorder); demyelinating core disorders, such as multiple sclerosis,acute transverse myelitis; disorders of the motor unit, such asneurogenic muscular atrophies (anterior horn cell degeneration, such asamyotrophic lateral sclerosis, infantile spinal muscular atrophy andjuvenile spinal muscular atrophy); Alzheimer's disease; Down's Syndromein middle age; Diffuse Lewy body disease; Senile Dementia of Lewy bodytype; Wemicke-Korsakoff syndrome; chronic alcoholism; Creutzfeldt-Jakobdisease; Subacute sclerosing panencephalitis, Hallerrorden-Spatzdisease; and Dementia pugilistica, or any subset thereof;

[0237] (E) malignant pathologies involving TNF-secreting tumors or othermalignancies involving TNF, such as, but not limited to leukemias(acute, chronic myelocytic, chronic lymphocytic and/or myelodyspasticsyndrome); lymphomas (Hodgkin's and non-Hodgkin's lymphomas, such asmalignant lymphomas (Burkitt's lymphoma or Mycosis fungoides));carcinomas (such as colon carcinoma) and metastases thereof;cancer-related angiogenesis; infantile haemangiomas;

[0238] (F) alcohol-induced hepatitis; and

[0239] (G) other diseases related to angiogenesis or VEGFNVPF, such asocular neovascularization, psoriasis, duodenal ulcers, angiogenesis ofthe female reproductive tract.

[0240] See, e.g., Berkow et al., eds., The Merck Manual, 16th edition,chapter 11, pp 1380-1529, Merck and Co., Rahway, N.J., 1992, whichreference, and references cited therein, are entirely incorporatedherein by reference. See also Folkman, Nature Medicine, Volume 1, No. 1(1995).

[0241] Such treatment comprises parenterally administering a single ormultiple doses of the antibody, fragment or derivative. Preferred forhuman pharmaceutical use are high affinity potent hTNFα-inhibitingand/or neutralizing murine and chimeric antibodies, fragments andregions of this invention.

[0242] Anti-TNF peptides or MAbs of the present invention can beadministered by any means that enables the active agent to reach theagent's site of action in the body of a mammal. In the case of theantibodies of this invention, the primary focus is the ability to reachand bind with TNF released by monocytes and macrophages or other TNFproducing cells. Because proteins are subject to being digested whenadministered orally, parenteral administration, i. e., intravenous,subcutaneous, intramuscular, would ordinarily be used to optimizeabsorption.

Therapeutic Administration

[0243] Anti-TNF peptides and/or MAbs of the present invention can beadministered either as individual therapeutic agents or in combinationwith other therapeutic agents. They can be administered alone, but aregenerally administered with a pharmaceutical carrier selected on thebasis of the chosen route of administration and standard pharmaceuticalpractice.

[0244] The dosage administered will, of course, vary depending uponknown factors such as the pharmacodynamic characteristics of theparticular agent, and its mode and route of administration; age, health,and weight of the recipient; nature and extent of symptoms, kind ofconcurrent treatment, frequency of treatment, and the effect desired.Usually a daily dosage of active ingredient can be about 0.01 to 100milligrams per kilogram of body weight. Ordinarily 1.0 to 5, andpreferably 1 to 10 milligrams per kilogram per day given in divideddoses 1 to 6 times a day or in sustained release form is effective toobtain desired results.

[0245] As a non-limiting example, treatment of TNF-related pathologieshumans or animals can be provided as a daily dosage of anti-TNFpeptides, monoclonal chimeric and/or murine antibodies of the presentinvention 0.1 to 100 mg/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, perday, on at least one of day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively, at least one ofweek 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19or 20, or any combination thereof, using single or divided doses ofevery 24, 12, 8, 6, 4, or 2 hours, or any combination thereof.

[0246] Since circulating concentrations of TNF tend to be extremely low,in the range of about 10 pg/ml in non-septic individuals, and reachingabout 50 pg/ml in septic patients and above 100 pg/ml in the sepsissyndrome (Hammerle, A. F. et al., 1989, infra) or can be only bedetectable at sites of TNF-mediated pathology, it is preferred to usehigh affinity and/or potent in vivo TNF-inhibiting and/or neutralizingantibodies, fragments or regions thereof, for both TNF immunoassays andtherapy of TNF-mediated pathology. Such antibodies, fragments, orregions, will preferably have an affinity for hTNFα, expressed as Ka, ofat least 10⁸ M⁻¹, more preferably, at least 10⁹ M⁻¹, such as10⁸−10¹⁰M⁻¹, 5×10⁸M⁻¹, 8×10⁸M⁻¹, 2×10⁹M⁻¹, 4×10⁹M⁻¹, 6×10⁹M⁻¹, 8×10⁹M⁻¹,or any range or value therein.

[0247] Preferred for human therapeutic use are high affinity murine andchimeric antibodies, and fragments, regions and derivatives havingpotent in vivo TNFα-inhibiting and/or neutralizing activity, accordingto the present invention, that block TNF-induced IL-6 secretion. Alsopreferred for human therapeutic uses are such high affinity murine andchimeric anti-TNFα antibodies, and fragments, regions and derivativesthereof, that block TNF-induced procoagulant activity, includingblocking of TNF-induced expression of cell adhesion molecules such asELAM-I and ICAM-I and blocking of TNF mitogenic activity, in vivo, insitu, and in vitro.

[0248] Dosage forms (composition) suitable for internal administrationgenerally contain from about 0.1 milligram to about 500 milligrams ofactive ingredient per unit. In these pharmaceutical compositions theactive ingredient will ordinarily be present in an amount of about0.5-95% by weight based on the total weight of the composition.

[0249] For parenteral administration, anti-TNF peptides or antibodiescan be formulated as a solution, suspension, emulsion or lyophilizedpowder in association with a pharmaceutically acceptable parenteralvehicle. Examples of such vehicles are water, saline, Ringer's solution,dextrose solution, and 5% human serum albumin. Liposomes and nonaqueousvehicles such as fixed oils can also be used. The vehicle or lyophilizedpowder can contain additives that maintain isotonicity (e.g., sodiumchloride, mannitol) and chemical stability (e.g., buffers andpreservatives). The formulation is sterilized by commonly usedtechniques.

[0250] Suitable pharmaceutical carriers are described in the most recentedition of Remington's Pharmaceutical Sciences, A. Osol, a standardreference text in this field of art.

[0251] For example, a parenteral composition suitable for administrationby injection is prepared by dissolving 1.5% by weight of activeingredient in 0.9% sodium chloride solution.

[0252] Anti-TNF peptides and/or antibodies of this invention can beadapted for therapeutic efficacy by virtue of their ability to mediateantibody-dependent cellular cytotoxicity (ADCC) and/orcomplement-dependent cytotoxicity (CDC) against cells having TNFassociated with their surface. For these activities, either anendogenous source or an exogenous source of effector cells (for ADCC) orcomplement components (for CDC) can be utilized. The murine and chimericantibodies, fragments and regions of this invention, their fragments,and derivatives can be used therapeutically as immunoconjugates (see forreview: Dilhman, R. O., Ann. Int. Med. 111:592-603 (1989)). Suchpeptides or Abs can be coupled to cytotoxic proteins, including, but notlimited to ricin-A, Pseudomonas toxin and Diphtheria toxin. Toxinsconjugated to antibodies or other ligands or peptides are well known inthe art (see, for example, Olsnes, S. et al., Immunol. Today 10:291-295(1989)). Plant and bacterial toxins typically kill cells by disruptingthe protein synthetic machinery.

[0253] Anti-TNF peptides and/or antibodies of this invention can beconjugated to additional types of therapeutic moieties including, butnot limited to, radionuclides, therapeutic agents, cytotoxic agents anddrugs. Examples of radionuclides which can be coupled to antibodies anddelivered in vivo to sites of antigen include ²¹²Bi , ¹³¹I, ¹⁸⁶Re, and⁹⁰Y, which list is not intended to be exhaustive. The radionuclidesexert their cytotoxic effect by locally irradiating the cells, leadingto various intracellular lesions, as is known in the art ofradiotherapy.

[0254] Cytotoxic drugs which can be conjugated to anti-TNF peptidesand/or antibodies and subsequently used for in vivo therapy include, butare not limited to, daunorubicin, doxorubicin, methotrexate, andMitomycin C. Cytotoxic drugs interfere with critical cellular processesincluding DNA, RNA, and protein synthesis. For a description of theseclasses of drugs which are well known in the art, and their mechanismsof action, see Goodman et al., Goodman and Gilman's THE PHARMACOLOGICALBASIS OF THERAPEUTICS, 8th Ed., Macmillan Publishing Co., 1990.

[0255] Anti-TNF peptides and/or antibodies of this invention can beadvantageously utilized in combination with other monoclonal or murineand chimeric antibodies, fragments and regions, or with lymphokines orhemopoietic growth factors, etc., which serve to increase the number oractivity of effector cells which interact with the antibodies.

[0256] Anti-TNF peptides and/or antibodies, fragments or derivatives ofthis invention can also be used in combination with TNF therapy to blockundesired side effects of TNF. Recent approaches to cancer therapy haveincluded direct administration of TNF to cancer patients orimmunotherapy of cancer patients with lymphokine activated killer (LAK)cells (Rosenberg et al., New Eng. J. Med. 313:1485-1492 (1985)) or tumorinfiltrating lymphocytes (TIL) (Kurnick et al. (Clin. Immunol.Immunopath. 38:367-380 (1986); Kradin et al., Cancer Immunol.Immunother. 24:76-85 (1987); Kradin et al., Transplant. Proc. 20:336-338(1988)). Trials are currently underway using modified LAK cells or TILwhich have been transfected with the TNF gene to produce large amountsof TNF. Such therapeutic approaches are likely to be associated with anumber of undesired side effects caused by the pleiotropic actions ofTNF as described herein and known in the related arts. According to thepresent invention, these side effects can be reduced by concurrenttreatment of a subject receiving TNF or cells producing large amounts ofTIL with the antibodies, fragments or derivatives of the presentinvention. Effective doses are as described above. The dose level willrequire adjustment according to the dose of TNF or TNF-producing cellsadministered, in order to block side effects without blocking the mainanti-tumor effect of TNF. One of ordinary skill in the art will know howto determine such doses without undue experimentation.

Treatment of Arthritis

[0257] In rheumatoid arthritis, the main presenting symptoms are pain,stiffness, swelling, and loss of function (Bennett JC. The etiology ofrheumatoid arthritis. In Textbook of Rheumatology (Kelley WN, Harris ED,Ruddy S, Sledge CB, eds.) WB Saunders, Philadelphia pp 879-886, 1985).The multitude of drugs used in controlling such symptoms seems largelyto reflect the fact that none is ideal. Although there have been manyyears of intense research into the biochemical, genetic,microbiological, and immunological aspects of rheumatoid arthritis, itspathogenesis is not completely understood, and none of the treatmentsclearly stop progression ofjoint destruction (Harris ED. RheumatoidArthritis: The clinical spectrum. In Textbook of Rheumatology (Kelley,et al., eds.) WB Saunders, Philadelphia pp 915-990, 1985).

[0258] TNFα is of major importance in the pathogenesis of rheumatoidarthritis. TNFα is present in rheumatoid arthritis joint tissues andsynovial fluid at the protein and mRNA level (Buchan G, et al., Clin.Exp. Immunol 73: 449-455, 1988), indicating local synthesis. Howeverdetecting TNFα in rheumatoid arthritis joints even in quantitiessufficient for bioactivation does not necessarily indicate that it isimportant in the pathogenesis of rheumatoid arthritis, nor that it is agood candidate therapeutic target. In order to address these questions,the effects of anti-TNF antibody and peptides (rabbit or monoclonal) onrheumatoid joint cell cultures, and for comparison, osteoarthritic cellcultures, have been studied. IL-1 production was abolished, showing TNFαas a suitable therapeutic target for the therapy of rheumatoidarthritis, since anti-TNFα blocks both TNF and IL-1, the two cytokinesknown to be involved in cartilage and bone destruction (Brennan et al.,Lancet 11:244-247, 1989).

[0259] Subsequent studies in rheumatoid arthritis tissues have supportedthis hypothesis. Anti-TNF Abs abrogated the production of anotherproinflammatory cytokine, GM-CSF (Haworth et al., Bur. J. Immunol.21:2575-2579, 1991). This observation has been independently confirmed(Alvaro-Gracia et al, 1991). It has also been demonstrated that anti-TNFdiminishes cell adhesion and HLA class II expression in rheumatoidarthritis joint cell cultures.

[0260] The administration of the antibody also established that VEGFNVPFserum levels were significantly decreased (FIG. 34) in rheumatoidarthritis (RA) patients. A prominant feature of rheumatoid arthritislesions is an infiltrate of inflammatory cells from the blood, togetherwith invading pannus which is associated with prominent new bloodformation, thus perpetuating the ingress of nutrients and cells, and theinflammatory reactions which culminate in bone and cartilagedestruction. VEGF is a potent inducer to angiogenesis and has beenimplicated in the formation of blood vessels and activation ofmicrovascular endothelium in RA.

[0261] VEGF serum levels are significantly increased in patients withactive RA, relative to serum levels in a population of age-matchedcontrols. Furthermore, a time-and dose-dependent decrease in serum VEGFlevels in RA patients after infusion with anti-TNFα, cA2, was observed.In patients who received 1 mg/kg cA2 (n=24), the maximal effect (33%decrease, p<0.001 versus change in placebo group and versus pre-infusionlevels) was detected 2 weeks post-infusion, but subsequently VEGF levelsreturned to pre-infusion levels. In contrast, patients who received 10mg/kg cA2 (n=21), the reduction was maintained even 4 weekspost-infusion (32% decrease, p<0.001), although serum levels were stillhigher than in normal individuals. The data indicate that treatment ofRA or other VEGF-mediated diseases with TNF antagonists decreases VEGFlevels in vivo, thereby leading to reduction in the vascularity of thepannus. Also, since VEGF is a survival factor for newly formed bloodvessels and endothelial cell apoptosis, as a consequence of suppressionof VEGF secretion, leads to vessel regression, TNF antagonists can exerta beneficial effect by causing regression of existing blood vessels inthe arthritic pannus.

Diagnostic Methods

[0262] The present invention also provides the above anti-TNF peptidesand antibodies, detectably labeled, as described below, for use indiagnostic methods for detecting TNFα in patients known to be orsuspected of having a TNFα-mediated condition.

[0263] Anti-TNF peptides and/or antibodies of the present invention areuseful for immunoassays which detect or quantitate TNF, or anti-TNFantibodies, in a sample. An immunoassay for TNF typically comprisesincubating a biological sample in the presence of a detectably labeledhigh affinity anti-TNF peptide and/or antibody of the present inventioncapable of selectively binding to TNF, and detecting the labeled peptideor antibody which is bound in a sample. Various clinical assayprocedures are well known in the art, e.g., as described in Immunoassaysfor the 80's, A. Voller et al., eds., University Park, 1981.

[0264] Thus, an anti-TNF peptide or antibody can be added tonitrocellulose, or another solid support which is capable ofimmobilizing cells, cell particles or soluble proteins. The support canthen be washed with suitable buffers followed by treatment with thedetectably labeled TNF-specific peptide or antibody. The solid phasesupport can then be washed with the buffer a second time to removeunbound peptide or antibody. The amount of bound label on the solidsupport can then be detected by known method steps.

[0265] By “solid phase support” or “carrier” is intended any supportcapable of binding peptide, antigen or antibody. Well-known supports orcarriers, include glass, polystyrene, polypropylene, polyethylene,dextran, nylon, amylases, natural and modified celluloses,polyacrylarnides, agaroses, and magnetite. The nature of the carrier canbe either soluble to some extent or insoluble for the purposes of thepresent invention. The support material can have virtually any possiblestructural configuration so long as the coupled molecule is capable ofbinding to TNF or an anti-TNF antibody. Thus, the support configurationcan be spherical, as in a bead, or cylindrical, as in the inside surfaceof a test tube, or the external surface of a rod. Alternatively, thesurface can be flat, such as a sheet, culture dish, test strip, etc.Preferred supports include polystyrene beads. Those skilled in the artwill know many other suitable carriers for binding antibody, peptide orantigen, or can ascertain the same by routine experimentation.

[0266] Well known method steps can determine binding activity of a givenlot of anti-TNF peptide and/or antibody. Those skilled in the art candetermine operative and optimal assay conditions by routineexperimentation.

[0267] Detectably labeling a TNF-specific peptide and/or antibody can beaccomplished by linking to an enzyme for use in an enzyme immunoassay(EIA), or enzyme-linked immunosorbent assay (ELISA). The linked enzymereacts with the exposed substrate to generate a chemical moiety whichcan be detected, for example, by spectrophotometric, fluorometric or byvisual means. Enzymes which can be used to detectably label theTNF-specific antibodies of the present invention include, but are notlimited to, malate dehydrogenase, staphylococcal nuclease,delta-5-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase.

[0268] By radioactively labeling the TNF-specific antibodies, it ispossible to detect TNF through the use of a radioimmunoassay (RIA) (see,for example, Work, et al., Laboratory Techniques and Biochemistry inMolecular Biology, North Holland Publishing Company, N.Y. (1978)). Theradioactive isotope can be detected by such means as the use of a gammacounter or a scintillation counter or by autoradiography. Isotopes whichare particularly useful for the purpose of the present invention are:³H, ¹²⁵I, ¹³¹I, ³⁵S, ¹⁴C, and, preferably, ¹²⁵I.

[0269] It is also possible to label the TNF-specific antibodies with afluorescent compound. When the fluorescent labeled antibody is exposedto light of the proper wave length, its presence can then be detecteddue to fluorescence. Among the most commonly used fluorescent labellingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

[0270] The TNF-specific antibodies can also be detectably labeled usingfluorescence-emitting met als such as ¹²⁵Eu, or others of the lanthanideseries. These met als can be attached to the TNF-specific antibody usingsuch met al chelating groups as diethylenetriaminepentaacetic acid(DTPA) or ethylenediamine-tetraacetic acid (EDTA).

[0271] The TNF-specific antibodies also can be detectably labeled bycoupling to a chemiluminescent compound. The presence of thechemiluminescently labeled antibody is then determined by detecting thepresence of luminescence that arises during the course of a chemicalreaction. Examples of particularly useful chemiluminescent labelingcompounds are luminol, isoluminol, theromatic acridinium ester,imidazole, acridinium salt and oxalate ester.

[0272] Likewise, a bioluminescent compound can be used to label theTNF-specific antibody, fragment or derivative of the present invention.Bioluminescence is a type of chemiluminescence found in biologicalsystems in which a catalytic protein increases the efficiency of thechemiluminescent reaction. The presence of a bioluminescent protein isdetermined by detecting the presence of luminescence. Importantbioluminescent compounds for purposes of labeling are luciferin,luciferase and aequorin.

[0273] Detection of the TNF-specific antibody, fragment or derivativecan be accomplished by a scintillation counter, for example, if thedetectable label is a radioactive gamma emitter, or by a fluorometer,for example, if the label is a fluorescent material. In the case of anenzyme label, the detection can be accomplished by colorometric methodswhich employ a substrate for the enzyme. Detection can also beaccomplished by visual comparison of the extent of enzymatic reaction ofa substrate in comparison with similarly prepared standards.

[0274] For the purposes of the present invention, the TNF which isdetected by the above assays can be present in a biological sample. Anysample containing TNF can be used. Preferably, the sample is abiological fluid such as, for example, blood, serum, lymph, urine,inflammatory exudate, cerebrospinal fluid, amniotic fluid, a tissueextract or homogenate, and the like. However, the invention is notlimited to assays using only these samples, it being possible for one ofordinary skill in the art to determine suitable conditions which allowthe use of other samples.

[0275] In situ detection can be accomplished by removing a histologicalspecimen from a patient, and providing the combination of labeledantibodies of the present invention to such a specimen. The antibody (orfragment) is preferably provided by applying or by overlaying thelabeled antibody (or fragment) to a biological sample. Through the useof such a procedure, it is possible to determine not only the presenceof TNF but also the distribution of TNF in the examined tissue. Usingthe present invention, those of ordinary skill will readily perceivethat any of a wide variety of histological methods (such as stainingprocedures) can be modified in order to achieve such in situ detection.

[0276] The antibody, fragment or derivative of the present invention canbe adapted for utilization in an immunometric assay, also known as a“two-site” or “sandwich” assay. In a typical immunometric assay, aquantity of unlabeled antibody (or fragment of antibody) is bound to asolid support that is insoluble in the fluid being tested and a quantityof detectably labeled soluble antibody is added to permit detectionand/or quantitation of the ternary complex formed between solid-phaseantibody, antigen, and labeled antibody.

[0277] Typical, and preferred, immunometric assays include “forward”assays in which the antibody bound to the solid phase is first contactedwith the sample being tested to extract the TNF from the sample byformation of a binary solid phase antibody-TNF complex. After a suitableincubation period, the solid support is washed to remove the residue ofthe fluid sample, including unreacted TNF, if any, and then contactedwith the solution containing a known quantity of labeled antibody (whichfunctions as a “reporter molecule”). After a second incubation period topermit the labeled antibody to complex with the TNF bound to the solidsupport through the unlabeled antibody, the solid support is washed asecond time to remove the unreacted labeled antibody. This type offorward sandwich assay can be a simple “yes/no” assay to determinewhether TNF is present or can be made quantitative by comparing themeasure of labeled antibody with that obtained for a standard samplecontaining known quantities of TNF. Such “two-site” or “sandwich” assaysare described by Wide (Radioimmune Assay Method, Kirkham, ed.,Livingstone, Edinburgh, 1970, pp. 199-206).

[0278] Other type of “sandwich” assays, which can also be useful withTNF, are the so-called “simultaneous” and “reverse” assays. Asimultaneous assay involves a single incubation step wherein theantibody bound to the solid support and labeled antibody are both addedto the sample being tested at the same time. After the incubation iscompleted, the solid support is washed to remove the residue of fluidsample and uncomplexed labeled antibody. The presence of labeledantibody associated with the solid support is then determined as itwould be in a conventional “forward” sandwich assay.

[0279] In the “reverse” assay, stepwise addition first of a solution oflabeled antibody to the fluid sample followed by the addition ofunlabeled antibody bound to a solid support after a suitable incubationperiod, is utilized. After a second incubation, the solid phase iswashed in conventional fashion to free it of the residue of the samplebeing tested and the solution of unreacted labeled antibody. Thedetermination of labeled antibody associated with a solid support isthen determined as in the “simultaneous” and “forward” assays. In oneembodiment, a combination of antibodies of the present inventionspecific for separate epitopes can be used to construct a sensitivethree-site immunoradiometric assay.

TNF Removal From Solutions

[0280] The murine and chimeric antibodies, fragments and regions,fragments, or derivatives of this invention, attached to a solidsupport, can be used to remove TNF from fluids or tissue or cellextracts. In a preferred embodiment, they are used to remove TNF fromblood or blood plasma products. In another preferred embodiment,themurine and chimeric antibodies, fragments and regions are advantageouslyused in extracorporeal immunoadsorbent devices, which are known in theart (see, for example, Seminars in Hematology, 26 (2 Suppl. 1) (1989)).Patient blood or other body fluid is exposed to the attached antibody,resulting in partial or complete removal of circulating TNF (free or inimmune complexes), following which the fluid is returned to the body.This immunoadsorption can be implemented in a continuous flowarrangement, with or without interposing a cell centrifugation step.See, for example, Terman, et al.,J. Immunol. 117:1971-1975 (1976).

[0281] Having now generally described the invention, the same will befurther understood by reference to certain specific examples which areincluded herein for purposes of illustration only and are not intendedto be limiting unless otherwise specified.

EXAMPLE I Production a Mouse Anti-Human TNF mAb

[0282] To facilitate clinical study of TNF mAb, a high-affinity potentinhibiting and/or neutralizing mouse anti-human TNF IgG1 mAb designatedA2 was produced.

[0283] Female BALB/c mice, 10 weeks old, were obtained from the JacksonLaboratory (Bar Harbor, Me.). Forty fig of purified E. coli-derivedrecombinant human TNF (rhTNF) emulsified with an equal volume ofcomplete Freund's adjuvant (obtained from Difco Laboratories) in 0.4 mlwas injected subcutaneously and intraperitoneally (i.p.) into a mouse.One week later, an injection of 5 μg of rhTNF in incomplete Freund'sadjuvant was given i.p. followed by four consecutive i.p. injections of10 μg of TNF without adjuvant. Eight weeks after the last injection, themouse was boosted i.p. with 10 μg ofTNF.

[0284] Four days later, the mouse was sacrificed, the spleen wasobtained and a spleen cell suspension was prepared. Spleen cells werefused with cells of the nonsecreting hybridoma, Sp2/0 (ATCC CRL1581), ata 4:1 ratio of spleen cells to Sp2/0 cells, in the presence of 0.3 ml of30% polyethylene glycol, PEG 1450. After incubation at 37° C. for 6hours, the fused cells were distributed in 0.2 ml aliquots into 96-wellplates at concentrations of 2×10⁴ SP2/0 cells per well. Feeder cells, inthe form of 5×10⁴ normal BALB/c spleen cells, were added to each well.

[0285] The growth medium used consisted of RPM1-1640 medium, 10%heat-inactivated fet al bovine serum (FBS) (HYCLONE), 0.1 mM minimumessential medium (MEM) nonessential amino acids, 1 mM sodium pyruvate, 2mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin (GIBCOLaboratories) and, for selection, hypoxanthine-aminopterin-thymidine(HAT) (Boehringer Mannheim). A solid-phase radioimmunoassay (RIA) wasemployed for screening supernatants for the presence of mAbs specificfor rhTNFα. This assay is described in Example II, below. The backgroundbinding in this assay was about 500 cpm. A supernatant was consideredpositive if it yielded binding of 2000 cpm or higher.

[0286] Of 322 supernatants screened, 25 were positive by RIA. Of these25, the one with the highest binding (4800 cpm) was designated A2.Positive wells were subcloned at limiting dilution on mouse feedercells. Upon further analysis of the supernatants in neutralizationassays, A2 was found to be the only positive clone showing potentinhibiting and/or neutralizing activity. Thus, the hybridoma line A2 wasselected. This line was maintained in RPM1-1640 medium with 10% FBS(GIBCO), 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 2 mML-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin.

[0287] Alternatively, anti-TNF antibodies which inhibit TNF biologicalactivity can be screened by binding to peptide including at least 5amino acids of residues 87-108 or both residues 59-80 and 87-108 of TNF(of SEQ ID NO: 1) or combinations of peptides contained therein, whichare used in place of the rTNF protein, as described above.

EXAMPLE II Characterization of an Anti-TNF Antibody of the PresentInvention Radioimmunoassays

[0288]E. coli-derived rhTNF was diluted to 1 μg/ml in BCB buffer, pH9.6, and 0.1 ml of the solution was added to each assay well. Afterincubation at 4° C. overnight, the wells were washed briefly with BCB,then sealed with 1% bovine incubated with 40 pg/ml of natural (GENZYME,Boston, MA) or recombinant (SUNTORY, Osaka, Japan) human TNFα withvarying concentrations of mAb A2 in the presence of 20 μg/mlcycloheximide at 39° C. overnight. Controls included medium alone ormedium+TNF in each well. Cell death was measured by staining withnaphthol blue-black, and the results read spectrophotometrically at 630nm. Absorbance at this wave length correlates with the number of livecells present.

[0289] It was found that A2 inhibited or neutralized the cytotoxiceffect of both natural and rhTNF in a dose-dependent manner (FIG. 3).

[0290] In another experiment, the specificity of this inhibiting and/orneutralizing activity was tested. A673/6 cells were seeded at 3×10⁴cells/well 20 hr before the TNF bioassay. Two-fold serial dilutions ofrhTNF, E. coli-derived recombinant human lymphotoxin (TNFβ ), and E.coli-derived recombinant murine TNF were prepared. The A2 hybridomasupernatant was added to an equal volume of the diluted TNFpreparations, and the mixtures were incubated at room temperature for 30min. Aliquots of 0.1 ml were transferred to the wells containing A673/6cells, 20 μg/ml of cycloheximide was added, and the cells were incubatedat 39° C. overnight. The cells were then fixed and stained forevaluation of cytotoxicity. The results indicate that mAb A2specifically inhibited or neutralized the cytotoxicity of rhTNFα,whereas it had no effect on human lymphotoxin (TNFO) (FIG. 4) or murineTNF (FIG. 5).

[0291] Experiments were next performed to analyze the cross-reactivityof mAb A2 with TNF derived from non-human primates. Monocytes isolatedfrom B514 (baboon), J91 (cynomolgus) and RH383 (rhesus) blood by Ficollgradient centrifugation and adherence, were incubated at 1×10⁵cells/well in RPMi 1640 medium with 5% FBS and 2 μg/ml of E. coli LPSfor 3 or 16 hr at 37° C. to induce TNF production. Supernatants fromduplicate wells were pooled and stored at 4° C. for less than 20 hruntil the TNF bioassay was performed, as described above, using A673/6cells. Two-fold dilutions of the culture supernatants were mixed witheither medium or purified mnAb A2 at a final concentration of 1 μg/ml,incubated at room temperature for 30 min. and aliquots transferred tothe indicator cells. The results showed that mAb A2 failed tosignificantly inhibit or neutralize the cytotoxic activity of TNFproduced by baboon, cynomolgus and rhesus monkey monocytes.

[0292] A further experiment was conducted with chimpanzee TNF. Monocytesisolated from CH563 (chimpanzee) blood were incubated as described aboveto generate TNF-containing supernatants. The ability of 10 μg/ml of mAbA2 to inhibit or neutralize the bioactivity of these supernatants wasassayed as above. Human TNF was used as a positive control. Results,shown in FIG. 6, indicate that mAb A2 had potent inhibiting and/orneutralizing activity for chimpanzee TNF, similar to that for human TNF(FIG. 7).

[0293] The inhibiting and/or neutralizing activity of mAb A2 wascompared with three other murine mAbs specific for human TNF, termedTNF-1, TNF-2 and TNF-3, and a control mAb. Two-fold serial dilutions ofpurified mAbs were mixed with rhTNF (40 pg/ml), incubated at roomtemperature for 30 min, and aliquots tested for TNF bioactivity asabove. It was found that mAbs TNF-1, TNF-2 and TNF-3 each had a similarmoderate degree of inhibiting and/or neutralizing activity. In contrast,mAb A2 had much more potent inhibiting and/or neutralizing activity.

EXAMPLE III General Strategy for Cloning Antibody V and C Genes

[0294] The strategy for cloning the V regions for the H and L chaingenes from the hybridoma A2, which secretes the anti-TNF antibodydescribed above, was based upon the linkage in the genome between the Vregion and the corresponding J (joining) region for functionallyrearranged (and expressed) Ig genes. J region DNA probes can be used toscreen genomic libraries to isolate DNA linked to the J regions.Although DNA in the genmline configuration (i.e., unrearranged) wouldalso hybridize to J. probes, this DNA would not be linked to a Ig Vregion sequence and can be identified by restriction enzyme analysis ofthe isolated clones.

[0295] The cloning utilized herein was to isolate V regions fromrearranged H and L chain genes using J_(H) and J_(K) probes. Theseclones were tested to see if their sequences were expressed in the A2hybridoma by Northern analysis. Those clones that contained expressedsequence were cloned into expression vectors containing human C regionsand transfected into mouse myeloma cells to determine if an antibody wasproduced. The antibody from producing cells was then tested for bindingspecificity and functionally compared to the A2 murine antibody.

EXAMPLE IV Construction of a L Chain Genomic Library

[0296] To isolate the L chain V region gene from the A2 hybridoma, asize-selected genomic library was constructed using the phage lambdavector charon 27. High molecular weight DNA was isolated from A2hybridoma cells and digested to completion with restriction endonucleaseHindIII. The DNA was then fractionated on a 0.8% agarose gel and the DNAfragments of three different size ranges of approximately 3 kb, 4 kb and6 kb were isolated from the gel by electroelution. The size ranges forlibrary construction were chosen based upon the size of HindIIIfragments that hybridized on a southern blot with the J_(K) probe. Afterphenol/chloroform extraction and ethanol precipitation, the DNAfragments from each size class were ligated with lambda charon 27 armsand packaged into phage particles in vitro using Gigapack Gold fromStratagene (LaJolla, Calif.).

[0297] These libraries were screened directly at a density ofapproximately 20,000 plaques per 150 mm petri dish using a ³²P-labeledJK probe. The mouse L chain J_(K) probe was a 2.7 kb HindIII fragmentcontaining all five JK segments. The probe was labeled with ³²P byrandom priming using a kit obtained from Boehringer Mannheim. Freenucleotides were removed by centrifugation through a Sephadex G-SOcolumn. The specific activities of the probe was approximately 10⁹cpm/μg.

[0298] Plaque hybridizations were carried out in 5x SSC, 50% formamide,2x Denhardt's reagent, and 200 μg/ml denatured salmon sperm DNA at 42°C. for 18-20 hours. Final washes were in 0.5x SSC, 0.1% SDS at 65° C.Positive clones were identified after autoradiography.

EXAMPLE V Construction of H Chain Genomic Library

[0299] To isolate the V region gene for the A2 H chain, a genomiclibrary was constructed in the lambda gt10 vector system. High molecularweight DNA was digested to completion with restriction endonucleaseEcoRI and fragments of approximately 7.5 kb were isolated after agarosegel electrophoresis. These fragments were ligated with lambda gt10 armsand packaged into phage particles in vitro using Gigapack Gold.

[0300] This library was screened at a density of 20,000 plaques per 150rnm plate using a JH probe. The JH probe was a 2kb BaniHI/EcoRi fragmentcontaining both J3 and J4 segments. The probe was labeled as in ExampleIII and had a similar specific radioactivity. Hybridization and washconditions were identical to those used in Example III.

EXAMPLE VI Cloning of the TNF-Specific V gene regions

[0301] Several positive clones were isolated from the H and L chainlibraries after screening approximately 10⁶plaques from each libraryusing the J_(H) and J_(K) probes, respectively. Following plaquepurification, bacteriophage DNA was isolated for each positive clone,digested with either EcoRI (H chain clones) or HindIII (L chain clones)and fractionated on 1% agarose gels. The DNA was transferred tonitrocellulose and the blots were hybridized with the J_(H) or the J_(K)probe.

[0302] Several H chain clones were obtained that contained 7.5 k/D EcoRIDNA encoding fragments of MAbs to the J_(H) probe. For the light chainlibraries, several clones from each of the three size-selected librarieswere isolated that contained HindEi fragments that hybridize to theJ_(K) probe. For the L chain, several independently derived Hindlfragments of 2.9 kb from the 2 kb library hybridized with a 1250 bp MRNAfrom A2, but not with SP2/0 MRNA (see Example VII). In addition, severalHindIII fragments derived from the 4 kb library hybridized both to theA2 mRNA and the fusion partner MRNA. A 5.7 kb HindIII fragment from the6 kb library did not hybridize to either RNA.

[0303] The observed lengths of hybridizing A2 MRNA were the correctsizes for H and L chain MRNA, respectively. Because the RNA expressionwas restricted to the A2 hybridoma, it was assumed that the 7.5 kb Hchain fragments and the 2.9 kb L chain fragments contained the correct Vregion sequences from A2. One example of each type was chosen forfurther study. The important functional test is the demonstration thatthese V regions sequences, when combined with appropriate C regionsequences, are capable of directing the synthesis of an antibody with aspecificity and affinity similar to that of the murine A2 antibody.

[0304] The 7.5 kb H chain fragment and the 2.9 kb L chain fragment weresubdloned into plasmid vectors that allow expression of the chimericmouse/human proteins in murine myeloma cells (see Examples VIII and IX).These plasmids were co-transfected into SP2/0 cells to ascertain ifintact antibody molecules were secreted, and if so, if they were of thecorrect specificity and affinity. Control transfections were alsoperformed pairing the putative anti-TNF H chain with an irrelevant, butexpressed, L chain; the putative anti-TNF L chain was also paired withan irrelevant, but expressed, H chain. The results indicated that the7.5 kb H chain fragment could be expressed, whereas the 2.9 kb L chainfragment could not. This was confirmed by DNA sequence analysis thatsuggested portions of the coding region were not in the proper aminoacid reading frame when compared to other known L chain amino acidsequences.

[0305] Because the 2.9 kb HindIII fragment appeared not to contain afunctional V gene, the 4.0 kb and 5.7 kb HindIII fragments isolated fromL chain libraries were cloned into expression vectors and tested forexpression of chimeric antibody after co-transfection with the 7.5 kb Hchain. The 5.7 kb HindIII fragment was incapable of supporting antibodyexpression, whereas the 4.0 kb HindIII fragment did support antibodyexpression. The antibody resulting from the co-transfection of the 7.5kb putative H chain V region and the 4.0 kb L chain V region waspurified, tested in solid phase TNF binding assay, and found to beinactive. It was concluded that the V region contained on the 4.0 kbHindIII fragment was not the correct anti-TNF V region, but wascontributed to the hybridoma by the fusion partner. This wassubsequently confirmed by sequence analysis of cDNA derived from the A2hybridoma and from the fuision partner.

[0306] Other independently derived L chain clones containing 2.9 kbHindIII fragments that hybridized with A2 MnRNA were characterized inmore detail. Although the restriction maps were similar, the clones fellinto two classes with respect to the presence or absence of an AccIenzyme site. The original (non-functional) 2.9 kb fragment (designatedclone 8.3) was missing an AccI site present in some other clones(represented by clone 4.3). The DNA sequence of clone 4.3 was extremelysimilar to clone 8.3, but contained a single amino acid reading framewith close homology to known L chains, unlike clone 8.3. The 2.9 kbHindIII fragment from clone 4.3 was subdloned into the L chainexpression vector and co-transfected with the putative anti-TNF H chaininto SP2/0 cells. An antibody was synthesized, purified and tested inthe solid phase TNF binding assay. This antibody bound to TNF, andtherefore, the clone 4.3 L chain V region was assumed to be the correctone.

[0307] The A2 murine hybridoma has been shown to contain at least fourrearranged L chain V region genes. At least two of these are expressedas proteins: clone 4.3 (the correct anti-TNF L chain gene) and the genecontained in the 4.0 kb HindIIIl fragment (contributed by the fusionpartner). The expression of two L chains implies that the resultingantibody secreted from the murine hybridoma is actually a mixture ofantibodies, some using the correct L chain, some using the incorrect Lchain, and some using one of each. The presence of two different Lchains in the murine A2 antibody has been confirmed by SDS gel andN-terminal protein sequence analysis of the purified antibody. Becauseconstruction of the chimeric A2 antibody involves cloning the individualH and L chain genes and expressing them in a non-producing cell line,the resulting antibody will have only the correct L chain and thereforeshould be a more potent antibody (see Examples X, XI and XII).

EXAMPLE VI Northern Analysis of Cloned DNA

[0308] Cloned DNA corresponding to the authentic H and L chain V regionsfrom the A2 hybridoma would be expected to hybridize to A2 MRNA.Non-functional DNA rearrangements at either the H or L chain geneticloci should not be expressed.

[0309] Ten μg total cellular RNA was subjected to electrophoresis on 1%agarose/formaldehyde gels (Sambrook et al., infra) and transferred tonitrocellulose. Blots were hybridized with random primed DNA probes in50% formamide, 2× Denhardt's solution, 5× SSC, and 200 μg/ml denaturedsalmon sperm DNA at 42° C. for 10 hours. Final wash conditions were 0.5×SSC, 0.1% SDS at 65° C.

[0310] The subcloned DNA fragments were labeled with ³²p by randompriming and hybridized to Northern blots containing total RNA derivedfrom A2 cells or from cells of SP2/0, the fusion partner parent of A2.The 7.5 kb EcoRI H chain fragment hybridized with a 2 kb mRNA from A2,but not with SP2/0 mRNA. Similarly, the 2.9 kb L chain Hindml fragment(clone 4.3) hybridized with a 1250 bp mRNA from A2, but not with SP2/0MRNA. The observed lengths of A2 MRNA hybridizing were the correct sizesfor H and L chain mRNA, respectively, confirming that the V regionsequences on these DNA fragments are expressed in A2 hybridoma cells.

EXAMPLE VIII Construction of Expression Vectors

[0311] The putative L (clone 4.3) and H chain V genes described abovewere joined to human kappa and gammal constant region genes inexpression vectors. The 7.5 kb EcoRI fragment corresponding to theputative V_(H) region gene from A2 was cloned into an expression vectorcontaining the human C_(gamma1) gene and the Ecogpt gene to yield theplasmid designated pA2HG1apgpt (see FIG. 8).

[0312] The 2.9 kb putative VL fragment from clone 4.3 was cloned into avector containing the human kappa C_(k) gene and the Ecogpt gene toallow selection in mammalian cells. The resulting plasmid was designatedpA2HuKapgpt (See FIG. 8).

EXAMPLE IX Expression of Chimeric Antibody Genes

[0313] To express the chimeric H and L chain genes, the expressionplasmids were transfected into cells of the non-producing mouse myelomacell line, SP2/0. Plasmid DNA to be transfected was purified bycentrifuging to equilibrium in ethidium bromide/cesium chloridegradients twice. Plasmid DNA (10-50 μg) was added to 10⁷ SP2/0 cells inmedium containing Hank's salts, and the mixture was placed in a BIORADelectroporation apparatus. Electroporation was performed at 20 volts,following which the cells were plated in 96 well microtiter plates.

[0314] Mycophenolic acid selection was applied after 24 hours and drugresistant colonies were identified after 1-2 weeks. Resistant colonieswere expanded to stable cell lines and tissue culture supernatant fromthese cell lines was tested for antibody using an ELISA assay with goatanti-human IgG Fc antibody and goat anti-human H+L conjugated withalkaline phosphatase (obtained from Jackson Laboratories).

[0315] The chimeric A2 antibody was purified from tissue culturesupernatant by Protein A-Sepharose chromatography. The supernatant wasadjusted to 0.1 M Tris, 0.002M EDTA, pH 8.0 and loaded on a ProteinA-Sepharose column equilibrated in the same buffer. The IgG was elutedwith 0.1 M citrate, pH 3.5, inhibited or neutralized with IM Tris, anddialyzed into phosphate buffered saline (PBS).

[0316] The purified chimeric antibody was evaluated for its binding andinhibiting and/or neutralizing activity.

EXAMPLE X Specificity of an Anti-TNF Chimeric Antibody

[0317] Since the antigen binding domain of cA2 was derived from murineA2, these mAbs would be expected to compete for the same binding site onTNF. Fixed concentrations of chimeric A2 and murine mAb A2 wereincubated with increasing concentrations of murine and chimeric A2competitor, respectively, in a 96-well microtiter plate coated withrhTNF (Dainippon, Osaka, Japan). Alkaline-phosphatase conjugatedanti-human immunoglobulin and anti-mouse immunoglobulin secondantibodies were used to detect the level of binding of chimeric andmurine A2, respectively. Cross-competition for TNF antigen was observedin this solid-phase ELISA format (FIGS. 9A and 9B). This finding isconsistent with the expected identical epitope specificity of cA2 andmurine A2.

[0318] The affinity constant for binding of mouse mAb A2 and cA2 torhTNFα was determined by Scatchard analysis (see, for example,Scatchard, Ann. N.Y. Acad. Sci. 51:660 (1949)). The results are shown inFIG. 10. This analysis involved measuring the direct binding of ¹²⁵Ilabelled cA2 to immobilized rhTNFα in a 96-well plate. The antibodieswere each labelled to a specific activity of about 9.7 μCi/μg by theiodogen method. An affinity constant (Ka) of 0.5×10⁹ liters/mole wascalculated for the mouse mAb A2. Unexpectedly, the chimeric A2 antibodyhad a higher affinity, with a Ka of 1.8×10⁹ liters/mole. Thus, thechimeric anti-TNFα antibody of the present invention was shown toexhibit a significantly higher affinity of binding to human TNFα thandid the parental murine A2 mAb. This finding was surprising, sincemurine and chimeric antibodies, fragments and regions would be expectedto have affinities that are equal to or less than that of the parentmAb.

[0319] Such high affinity anti-TNF antibodies, having affinities ofbinding to TNFα of at least 1×10⁸M⁻¹, more preferably at least 1×10⁹ M⁻¹(expressed as Ka) are preferred for immunoassays which detect very lowlevels of TNF in biological fluids. In addition, anti-TNF antibodieshaving such high affinities are preferred for therapy of TNF-α-mediatedconditions or pathology states.

[0320] The specificity of cA2 for TNF was confirmed by testing forcross-neutralization of human lymphotoxin (TNF-β). Lymphotoxin sharessome sequence homology and certain biological activities, for example,tumor cell cytotoxicity, with TNF (Pennica, et al., Nature 312:724-729(1984)). Cultured human A673 cells were incubated with increasingconcentrations of human lymphotoxin (GENENTECH₁ San Francisco, Calif.)with or without 4 μg/ml chimeric A2 in the presence of 20 μg/mlcycloheximide at 39C overnight. Cell death was measured by vitalstaining with naphthol blue-black, as above. The results indicated thatcA2 was ineffective at inhibiting and/or neutralizing human lymphotoxin,confirming the TNFα-specificity of the chimeric antibody.

[0321] The ability of A2 or cA2 to react with TNF from different animalspecies was also evaluated. As mentioned earlier, there are multipleepitopes on human TNF to which inhibiting and/or neutralizing mAbs willbind (Moller, et al., infra). Human TNF has bioactivity in a wide rangeof host animal species. However, certain inhibiting and/or neutralizingepitopes on human TNF are conserved amongst different animal species andothers appear to be restricted to humans and chimpanzees.

[0322] Neutralization experiments utilized endotoxin-activated cellsupernatants from freshly isolated human, chimpanzee, rhesus andcynomolgus monkey, baboon, pig, dog, rabbit, or rat monocytes as the TNFsource. As discussed above, murine mnAb A2 inhibited or neutralizedactivity of only human and chimpanzee TNF, and had no effect on TNFderived from other primates and lower animals. A2 also did not inhibitor neutralize the cytotoxic effect of recombinant mouse TNF.

[0323] Thus, the epitope recognized by A2 is one shared by human andchimpanzee TNFα. Chimeric A2 was also tested in this manner forcross-reactivity with monocyte-derived TNF from rat, rabbit, dog andpig, as well as with purified recombinant mouse TNFα, and natural andrecombinant human TNFα. Chimeric A2 only inhibited or neutralizednatural and recombinant human TNFα. Therefore, cA2 appears to sharespecies specificity with murine A2.

EXAMPLE XI In Vitro Activity and Neutralization Efficacy of a ChimericAnti-TNF Antibody

[0324] Both the murine and chimeric anti-TNFα antibodies, A2 and cA2were determined to have potent TNF-inhibiting and/or neutralizingactivity. In the TNF cytotoxicity assay described above, murine A2, at aconcentration of about 125 ng/ml completely inhibited or neutralized thebiological activity of a 40 pg/ml challenge of TNFα. Two separatedeterminations of inhibiting and/or neutralizing potency, expressed asthe 50% Inhibitory Dose (ID50) were determined to be 15.9±1.01 and17.9±1.6 ng/ml (Mean+Std error). Thus the mAb A2 has an ID50 of about 17ng/ml.

[0325] In this same experimental system, three other murine anti-TNFαantibodies (termed TNF-1, TNF-2 and TNF-3) of comparable bindingaffinity to TNF were found to have ID50 values of 1-2 orders ofmagnitude greater, and thus were significantly less potent inneutralization than A2.

[0326] The ability of cA2 to inhibit or neutralize human TNFαbioactivity in vitro was tested using the bioassay system describedabove. Cultured A673 cells were incubated with 40 pg/ml natural(Genzyme, Boston, Mass.) or recombinant (Suntory, Osaka, Japan) humanTNF with or without antibody overnight as above, and cell death wasmeasured by vital staining. As expected based upon the above resultswith the A2 mouse mAb, cA2 also inhibited or neutralized both naturaland rhTNF in a dose-dependent manner in the cytotoxicity assay (FIG.11). In this assay format, levels of cA2 as low as 125 ng/ml completelyabolished the toxic activity of TNF. Upon repeated analysis, the cA2exhibited greater TNF-inhibiting and/or neutralizing activity than didthe parent murine A2 mAb. Such inhibiting and/or neutralizing potency,at antibody levels below 1 μg/ml, can easily be attained in the blood ofa subject to whom the antibody is administered. Accordingly, such highlypotent inhibiting and/or neutralizing anti-TNF antibodies, in particularthe chimeric antibody, are preferred for therapeutic use inTNFα-mediated pathologies or conditions.

[0327] As mentioned above, TNF induces cellular secretion of IL-6.Furthermore, there is evidence that IL-6 is involved in thepathophysiology of sepsis, although the precise role of IL-6 in thatsyndrome is unclear (Fong, et al.,J. Exp. Med. 170:1627-1633 (1989);Starnes Jr., et al.,J. Immunol. 145:4185-4191 (1990)). The ability ofcA2 to inhibit or neutralize TNF-induced IL-6 secretion was evaluatedusing cultured human diploid FS-4 fibroblasts. The results in Table 2show that cA2 was effective in blocking IL-6 secretion in cells that hadbeen incubated overnight with TNF. TNF-induced IL-6 secretion was notinhibited in the absence of a mAb or in the presence of a control mAbspecific for an irrelevant antigen. TABLE 2 In Vitro Neutralization ofTNF-Induced IL-6 Secretion TNF Concentration (ng/ml) Antibody 0 0.3 1.57.5 None <0.20 1.36 2.00 2.56 Control mAb <0.20 1.60 1.96 2.16 cA2 <0.20<0.20 <0.20 0.30

[0328] The ability of TNF to activate procoagulant and adhesion moleculeactivities of endothelial cells (EC) is thought to be an importantcomponent of pathology pathophysiology. In particular, this can beassociated with the vascular damage, disseminated intravascularcoagulation, and severe hypotension that is associated with the sepsissyndrome. Therefore, the ability of cA2 to block TNF-induced activationof cultured human umbilical vein endothelial cells (HUVEC) wasevaluated. TNF stimulation of procoagulant activity was determined byexposing intact cultured HUVEC cells to TNF (with or without antibody)for 4 hours and analyzing a cell lysate in a human plasma clottingassay. The results in Table 3 show the expected upregulation by TNF ofHUVEC procoagulant activity (reflected by a decreased clotting time).Chimeric antibody cA2 effectively inhibited or neutralized this TNFactivity in a dose-dependent manner. TABLE 3 In Vitro Neutralization ofTNF-Induced Procoagulant Activity TNF Concentration (ng/ml) Antibodyμg/ml 250 25 0 None —  64 ± 4*  63 ± 1 133 ± 13 Control Ab 10.00  74 ± 6N.D. 178 ± 55 cA2 10.00 114 ± 5  185 ± 61 141 ± 18 cA2 3.30 113 ± 2 147± 3 N.D. cA2 1.10 106 ± 1 145 ± 8 N.D. A2 0.37  73 ± 17 153 ± 4 N.D. cA20.12  64 ± 1  118 ± 13 N.D.

[0329] In addition to stimulating procoagulant activity, TNF alsoinduces surface expression of endothelial cell adhesion molecules suchas ELAM-1 and ICAM-1. The ability of cA2 to inhibit or neutralize thisactivity of TNF was measured using an ELAM-1 specific detectionradioimmunoassay. Cultured HUVEC were stimulated with 250 ng/ml TNF(Dainippon, Osaka, Japan) with or without antibody at 37° C. overnightin a 96-well plate format. Surface expression of ELAM-1 was determinedby sequential addition of a mouse anti-human ELAM-1 mAb and¹²⁵l-labelled rabbit anti-mouse immunoglobulin second antibody directlyto culture plates at 4° C.

[0330] As shown in FIG. 12, TNF induced the expression of ELAM-1 on thesurface of cultured HUvEC cells, and this activity was again effectivelyblocked in a dose-related manner by cA2.

[0331] Finally, TNF is known to stimulate mitogenic activity in culturedfibroblasts. Chimeric A2 inhibited or neutralized TNF-inducedmitogenesis of human diploid FS-4 fibroblasts cultures, confirming thepotent inhibiting and/or neutralizing capability of cA2 against a broadspectrum of in vitro TNF biological activities.

EXAMPLE XII Determination of Amino Acid Sequences (Epitope) on HumanTNF-α Recognized by cA2 mAb Reagents

[0332] The following reagents are readily available from commercialsources. FMOC—L—Ala—OPfp, FMOC—L—Cys(Trt)—OPfp, FMOC—L—Asp(OtBu)—OPfp,FMOC—L—Giu (OtBu)—OPfp, FMOC—L—Phe—OPfp, FMOC—Gly—OPfp, FMOC—L—His(Boc)—OPfp, FMOC—L—Ile—OPfp, FMOC—L—Lys(Boc)—OPfp, FMOC—L—Leu—OPfp,FMOC—L—Asn—OPfp, FMOC—L—Pro—OPfp, FMOC—L—Gin—OPfp, FMOC—L—Arg(Mtr)—OPfp,FMOC—L—Ser(tBu)—ODhbt, FMOC—L—Thr(tBu)—ODhbt, FMOC—L—Val—OPfp,FMOC—L—Trp—OPfp, FMOC—L—Try(tBu)—OPfp, and 1-hydrox-fbenotriazol (HOBT)were obtained from Cambridge Research Biochemicals. Piperidine and wasobtained from Applied Biosystems, Inc. 1-Methyl-2-Pyrrolidinone (NMP)was obtained from EM Science; Methanol from JT Baker; Acetic Anhydridefrom Applied Biosystems, Inc., Trifluoroaccetic acid (TFA) from AppliedBiosystems, Inc.; Diisopropylamne (DIEA), Triethylamine, Dithiothreitol(DTT) and Anisole from Aldrich and Hydrochloric Acid (HCI) from JTBaker.

[0333] Abbreviations: FMOC, 9-fluorenylmethoxycarbonyl; tBu t-butylether; OrB, t-butyl ester; Boc, t-butyloxycarbonyl; Mtr,4-methoxy-2,3,6-trimethyl- benzenesulfonyl; Trt, trityl; OPfp,pentafluorophenylester; ODnbt. oxo-benzotriazone ester.

[0334] A chimeric antibody of the present invention, designated cA2, wasused to determine which portions of the TNF amino acid sequence wereinvolved in inhibitory binding by the antibody by epitope mapping,whereby the amino acid sequences of TNF-α recognized by cA2 have beenidentified.

[0335] The complete primary sequence of human TNFα, according to Pennicaet al., Nature 312:724-729 (1984) is shown in FIG. 13 (SEQ ID NO:1).Overlapping decapeptides beginning with every second amino acid andcovering the entire amino acid sequence of human TNF-α were synthesizedon polyethylene pins using the method of Gysen (Gysen et al., Peptides:Chemistry and Biological, Proceedings of the Twelfth American PeptideSymposium, p. 519-523, Ed, G. R. Marshall, Escom, Leiden, 1988). Sets ofpeptide pins bearing free N-terminal amino groups and acetylatedN-terminal amino groups were individually prepared. Both sets of peptidepins were incubated in solutions containing the anti-TNF mAb cA2 todetermine the amino acid sequences that make up the cA2 epitope on humanTNF-α, as described below. FIG. 14A shows the results of binding to theoverlapping decapeptides that comprise the entire sequence of humanTNFα. The O.D. (optional density) correlates directly with the increaseddegree of cA2 binding. FIG. 14B shows the results of binding of cA2 tothe same set of peptide pins in the presence of human TNFα. Thiscompetitive binding study delineates peptides which can shownon-specific binding to CA2.

[0336] There are at least two non-contiguous peptide sequences of TNF-αrecognized by cA2. Using the conventional protein numbering systemwherein the N-terminal amino acid is number 1, the cA2 mAb recognizes anepitope composed at least in part of amino acids located within residues87-108 or both residues 59-80 and 87-108 of TNF (SEQ ID NO:1). FIG. 15presents these non-contiguous sequences within the TNF sequence.

[0337] Unexpectedly, the mAb cA2 blocks the action of TNF-α withoutbinding to the putative receptor binding locus, which can include one ormore of, e.g., 11-13, 37-42, 49-57 or 155-157 of hTNFα (of SEQ ID NO:1). Preferred anti-TNF mAbs are those that inhibit this binding of humanTNF-α to its receptors by virtue of their ability to bind to one or moreof these peptide sequences. These antibodies can block the activity ofTNF by virtue of binding to the cA2 epitope, such binding demonstratedto inhibit TNF activity. The identification of those peptide sequencesrecognized by cA2 provides the information necessary to generateadditional MAbs with binding characteristics and therapeutic utilitythat parallel the embodiments of this application.

Peptide Pin Synthesis

[0338] Using an epitope mapping kit purchased from Cambridge ResearchBiochemicals, Inc. (CRB), dodecapeptides corresponding to the entiresequence of human TNF-α were synthesized on polyethylene pins.

[0339] A synthesis schedule was generated using the CRB epitope mappingsoftware. Prior to the first amino acid coupling, the pins weredeprotected with a 20% piperidine in NMP solution for 30 minutes at roomtemperature. After deprotection, the pins were washed with NMP for fiveminutes at room temperature, followed by three methanol washes.Following the wash steps, the pins were allowed to air dry for at least10 minutes.

[0340] The following procedure was performed for each coupling cycle:

[0341] 1) The amino acid derivatives and the HOBT were weighted outaccording to the weights required in the synthesis schedule.

[0342] 2) The HOBT was dissolved in the appropriate amount of NMPaccording to the synthesis schedule.

[0343] 3) The amino acid derivatives were dissolved in the recommendedamount of HOBT solution and 150 microliters were pipeted into theappropriate wells as directed by the well position sheet of thesynthesis schedule.

[0344] 4) The blocks containing the pins were placed into the wells, andthe “sandwich” units stored in plastic bags in a 30° C. water bath for18 hours.

[0345] 5) The pins were removed from the wells and washed once (for 5minutes) with NMP, three times (for two minutes) with methanoi and airdried for 10 minutes.

[0346] 6) The pins were deprotected as described above and the procedurerepeated.

[0347] To acetylate the peptides on one block of pins, the peptide pinswere washed, deprotected and treated with 150 microliters of a solutioncontaining NMP; acetic anhydride:triethylamine (5:2:1) for 90 minutes at30° C., followed by the washing procedure outlined above. The second setof peptide pins was deprotected by not acetylated to give freeN-terminal amino groups.

[0348] The final deprotection of the peptides to remove the side chainprotecting groups was done using a mixture ofTFA:anisole:dithiothreitol, 95:2.5:2.5 (v/v/w) for four hours at ambienttemperature. After deprotection, the pins were air dried for 10 minutes,followed by a 15 minute sonication in a solution of 0.1% HCl inmethanol/distilled water (1:1). The pins dried over night and were thenready for testing.

ELISA Assay for cA2 Binding to TNF-α Peptide PINs Reagents: DisruptionBuffer

[0349] Sodium dihydrogen phosphate (31.2 g, Sigma cat # S-0751 orequivalent) and sodium dodecylsulfate (20.0 g, Sigma cat # L-3771 orequivalent) were dissolved in 2.0 L of milliQ water. The pH was adjustedto 7.2±0.1 with 50% w/w sodium hydroxide (VWR cat # VW6730-3 orequivalent).

Blocking Buffer

[0350] Sodium dihydrogen phosphate (0.39 g, Sigma cat #S-0751 orequivalent) disodium hydrogen phosphate (1.07 g, Baker cat # 3828-1 orequivalent) and sodium chloride (8.50 g, Baker cat # 3624-5 orequivalent) were dissolved in 1.0 L of milliQ water. The pH was adjustedto 7.2±0.1 with 50% w/w sodium hydroxide (VWR cat VW6730-3 orequivalent). Chicken egg albumin (10.0 g, Sigma cat #A-5503 orequivalent) and bovine serum albumin (10.0 g, Sigma, cat #A-3294 orequivalent) were dissolved at room temperature with gentle stirring. Thesolution was filtered, and to the solution was added Tween 20 (2.0 ml,Sigma cat #P-13.79 or equivalent). The solution was stirred gently atroom temperature for 30 min, filtered and stored at 40°.

PBS/Tween 20

[0351] A 10× concentrate was prepared by dissolving sodium dihydrogenphosphate (3.90 g, Sigma cat # S-0751 or equivalent), disodium hydrogenphosphate (10.70 g, Baker cat #3828-1 or equivalent) and sodium chloride(85.0 g, Baker cat #3624-5 or equivalent) in 1.0 L of milliQ water. ThepH was adjusted to 7.2±0.1 with 50% w/w sodium hydroxide (VWR cat #VW6730 or equivalent). To the solution was added Tween 20 (5.0 mL, Sigmacat #P-1379 or equivalent), and the mixture stirred gently. Just priorto use 100 mL of this solution was diluted to 1.0 L with milliQ water.

Substrate solution

[0352] Substrate buffer was prepared by dissolving citric acid (4.20 g,Malinckrodt cat #0627 or equivalent) and disodium hydrogen phosphate(7.10 g, Baker cat #3828-1 or equivalent) in 1.0 L of milliQ water. ThepH was adjusted to 5.00 with 50% w/w sodium hydroxide (VWR cat #VW6730-3or equivalent). Immediately prior to use an OPD substrate tablet (30 mg,Sigma cat #P-8412 or equivalent and 30% (v/v) hydrogen peroxide (40 μL,Sigma cat #P-1379 or equivalent) were added to the substrate buffer 25.0mL). The solution was wrapped in foil and mixed thoroughly.

4 NH₂S04

[0353] Sulfuric acid (53 mL, EM Science cat #SX1244-5 or equivalent) wasslowly added to MILLI-Q water (447 mL) and cooled to room temperatureprior to use.

Equipment

[0354] Molecular Devices Model nu-max plate reader or equivalent.Scientific Products Model R4140 Oscillating table shaker and equivalent.BRANSON Model 5200 ultra-sonic bath or equivalent. FINNPIPETTE Model4172317 multichannel pipeter or equivalent. CORNING Model 25801 96 welldisposable polystyrene Elisa Plates.

[0355] Prior to use and after each subsequent use the peptide pins werecleaned using the following procedure. Disruption buffer (2.0 L) washeated to 600 and placed in an ultra-sonic bath in a fume hood. To thedisruption buffer was added dithiolthreitol (2.5 g, Sigma cat #D-0632 orequivalent). The peptide pins were sonicated in this medium for 30 min,washed thoroughly with milliQ waster, suspended in a boiling ethanolbath for 2 min, and air-dried.

[0356] Blocking buffer (200 μL) was added to a 96 well disposablepolystyrene Elisa plate and the peptide pins suspended in the wells. Thepeptide pins and plate were incubated for 2 hours at room temperature onan oscillating table shaker. The plates and peptide pins were washedwith PBS/Tween 20 (four times). To each well was added a 20 μg/mlconcentration of cA2 antibody (diluted with blocking buffer, 175μL/well). TNF competition was done by incubation of TNFα (40 μg/ml) andcA2 (20 μg/ml) in BSA/ovalbumin/ BBS for three hours at roomtemperature. The peptide pins were suspended in the plate and incubatedat 4° overnight. The peptide pins and plate were washed with PBS/Tween20 (four times). To each well was added anti-human goat antibodyconjugated to horseradish peroxidase (diluted with blocking buffer to{fraction (1/2000)}, 175 μL/well, Jackson IMMUNORESEARCH Labs). Thepeptide pins were suspended in the plate, and incubated for 1 hour atroom temperature on a oscillating table shaker. The plates and peptidepins were washed with PBS/Tween 20 (four times). To each well was addedfreshly prepared substrate solution (150 μL/well), the peptide pins weresuspended in the plate and incubated for 1 hour at room temperature onan oscillating table shaker. The peptide pins were removed and to eachwell is added 4N H₂S0₄ (50 μL). The plates were read in a MolecularDevices plate reader (490 nm, subtracting 650 nm as a blank), and theresults are shown in FIGS. 14A and 14B, as described above.

EXAMPLE XIII Production of Mouse Anti-Human TNF mAb Using TNF PeptideFragments

[0357] Female BALB/c mice, as in Example I above, are injectedsubcutaneously and intraperitoneally (i.p.) with forty μg of purified E.coli-derived recombinant human TNF (TNF) fragments comprising anti-TNFepitopes of at least 5 amino acids located within the non-contiguoussequence 59-80, 87-108 or both residues 59-80 and 87-108 of TNF (of SEQID NO: 1), as presented above, emulsified with an equal volume ofcomplete Freund's adjuvant (obtained from Difco Laboratories) in 0.4 mlis into a mouse. One week later, a booster injection of 5 μg of theseTNF fragments in incomplete Freund's adjuvant is given i.p. followed byfour consecutive i.p. injections of 10 μg of TNF fragments includinganti-TNF epitopes including amino acids from residues 59-80, 87-108 orboth 59-80 and 87-108 of hTNFα (of SEQ ID NO: 1) without adjuvant. Eightweeks after the last injection, the mouse is boosted i.p. with 10 μg ofTNF.

[0358] Four days later, the mouse is sacrificed, the spleen is obtainedand a spleen cell suspension is prepared. Spleen cells are fused withcells of the nonsecreting hybridoma, Sp2/0 (ATCC CRL1581), at a 4:1ratio of spleen cells to Sp2/0 cells, in the presence of 0.3 ml of 30%polyethylene glycol, PEG 1450. After incubation at 37° C. for 6 hours,the fused cells are distributed in 0.2 ml aliquots into 96-well platesat concentrations of 2×10⁴ SP2/0 cells per well. Feeder cells, in theform of 5×10⁴ normal BALB/c spleen cells, are added to each well.

[0359] The growth medium used consisted of RPM1-1640 medium, 10%heat-inactivated fet al bovine serum (FBS) (Hyclone), 0.1 mM MEMnonessential amino acids, 1 mM sodium pyruvate, 2 mM L-glutamine, 100U/ml penicillin, 100 μg/ml streptomycin (GIBCO Laboratories) and, forselection, hypoxanthine-aminopterin-thymidine (HAT) (BoehringerMannheim). A solid-phase radioimmunoassay (RIA) is employed forscreening supernatants for the presence of mAbs specific for TNFαfragments including portions of residues 59-80, 87-108 or both 59-80 and87-108 of hTNFα (of SEQ ID NO:1). This assay is described in Example II,above. The background binding in this assay is about 500 cpm. Asupernatant is considered positive if it yielded binding of 2000 cpm orhigher.

[0360] Of the supernatants screened, one or more positive supernatantsare routinely identified by RIA. Of these positive supernatants, thehighest binding (as shown by the higher cpm values) are subcloned atlimiting dilution on mouse feeder cells. Upon further analysis of thesupernatants in neutralization assays, routinely one or more antibodiesare found to have potent inhibiting and/or neutralizing activity. Thesepositive and inhibiting and/or neutralizing hybridoma lines are thenselected and maintained in RPM1-1640 medium with 10% FBS (GIBCO), 0.1 mMnonessential amino acids, 1 mM sodium pyruvate, 2 mM L-glutamine, 100U/ml penicillin and 100 μg/ml streptomycin.

EXAMPLE XIV Production of Murine and Chimeric Antibodies, Fragments andRegions from TNF Peptides

[0361] Murine and chimeric antibodies, fragments and regions areobtained by construction of chimeric expression vectors encoding themouse variable region of antibodies obtained in Example XIII and humanconstant regions, as presented in Examples IV-IX above.

[0362] The resulting chimeric A2 antibody is purified from tissueculture supernatant by Protein A-Sepharose chromatography. Thesupernatant is adjusted to 0.1 M Tris, 0.002M EDTA, pH 8.0 and loaded ona Protein A-Sepharose column equilibrated in the same buffer. The IgG isthen eluted with 0.1M citrate, pH 3.5, neutralized with 1M Tris, anddialyzed into phosphate buffered saline (PBS).

[0363] The purified murine and chimeric antibodies, fragments andregions are evaluated for its binding and inhibiting and/or neutralizingactivity.

EXAMPLE XV In Vitro Activity and Neutralization Efficacy of a ChimericAnti-TNF Antibody

[0364] Both the murine and chimeric anti-TNFα antibodies of the presentinvention, as obtained according to Examples XIII and XIV, aredetermined to have potent TNF-inhibiting and/or neutralizing activity,as shown for example, in the TNF cytotoxicity assay described above,expressed as the 50% Inhibitory Dose (ID50).

[0365] In this same experimental system, three other murine anti-TNFαantibodies (termed TNF-1, TNF-2 and TNF-3) of comparable bindingaffinity to TNF are found to have ID50 values of 1-2 orders of magnitudegreater, and thus have significantly less potency in neutralization,than both the murine and chimeric anti-TNFα antibodies of the presentinvention.

[0366] The ability of both the murine and chimeric anti-TNFα antibodiesof the present invention, as obtained according to Examples XIII andXIV, to inhibit or neutralize human TNFα bioactivity in vitro is testedusing the bioassay system described above. Cultured cells producing themurine or chimeric anti-TNFα antibodies of the present invention, asobtained according to Examples XIII and XIV, are incubated with 40 pg/mlnatural (Genzyme, Boston, Mass.) or recombinant (Suntory, Osaka, Japan)human TNF with or without antibody overnight as above, and cell death ismeasured by vital staining. As expected, both the murine and chimericanti-TNFα antibodies of the present invention, as obtained according toExamples XIII and XIV, inhibited or neutralized both natural and TNF ina dose-dependent manner in the cytotoxicity assay. Such inhibitingand/or neutralizing potency, at antibody levels below 1 μg/ml, caneasily be attained in the blood of a subject to whom the antibody isadministered. Accordingly, such highly potent inhibiting and/orneutralizing anti-TNF antibodies, in particular the chimeric antibody,are preferred for therapeutic use in TNFα-mediated pathologies orconditions.

[0367] The ability of cA2 to inhibit or neutralize TNF-induced IL-6secretion is evaluated using cultured human diploid FS-4 fibroblasts.The results are expected to show that both murine and chimeric anti-TNFαantibodies of the present invention, as obtained according to ExamplesXIII and XIV, are effective in blocking IL-6 secretion in cells that hadbeen incubated overnight with TNF. TNF-induced IL-6 secretion is notinhibited in the absence of a mAb or in the presence of a control mAbspecific for an irrelevant antigen.

[0368] The ability of TNF to activate procoagulant and adhesion moleculeactivities of endothelial cells (EC) is thought to be an importantcomponent of pathology pathophysiology. In particular, this can beassociated with the vascular damage, disseminated intravascularcoagulation, and severe hypotension that is associated with the sepsissyndrome. Therefore, the ability of both the murine and chimericanti-TNFα antibodies of the present invention, as obtained according toExamples XIII and XIV, to block TNF-induced activation of cultured humanumbilical vein endothelial cells (HUVEC) is evaluated. TNF stimulationof procoagulant activity is determined by exposing intact cultured HUVECcells to TNF (with or without antibody) for 4 hours and analyzing a celllysate in a human plasma clotting assay. The results are expected toshow the expected upregulation by TNF of HUVEC procoagulant activity(reflected by a decreased clotting time). Both the murine and chimericanti-TNFα antibodies of the present invention, as obtained according toExamples XIII and XIV, are expected to effectively inhibit or neutralizethis TNF activity in a dose-dependent manner.

[0369] In addition to stimulating procoagulant activity, TNF alsoinduces surface expression of endothelial cell adhesion molecules suchas ELAM-1 and ICAM-1. Both the murine and chimeric anti-TNFα antibodiesof the present invention, as obtained according to Examples XIII andXIV, are expected to inhibit or neutralize this activity of TNF ismeasured using an ELAM-1 specific detection radioimmunoassay. CulturedHUVEC are stimulated with 250 ng/ml TNF (Dainippon, Osaka, Japan) withor without antibody at 37° C. overnight in a 96-well plate format.Surface expression of ELAM-1 is determined by sequential addition of amouse anti-human ELAM-1 mAb and ¹²⁵I-labelled rabbit anti-mouseimmunoglobulin second antibody directly to culture plates at 4° C.

[0370] TNF is expected to induce the expression of ELAM-1 on the surfaceof cultured HUVEC cells, and this activity is again expected to beeffectively blocked in a dose-related manner by both the murine andchimeric anti-TNFα antibodies of the present invention, as obtainedaccording to Examples XIII and XIV.

[0371] Finally, TNF is known to stimulate mitogenic activity in culturedfibroblasts. Both the murine and chimeric anti-TNFα antibodies of thepresent invention, as obtained according to Examples XIII and XIV, areexpected to inhibit or neutralize TNF-induced mitogenesis of humandiploid FS-4 fibroblasts cultures, confirming the potent inhibitingand/or neutralizing capability of both the murine and chimeric anti-TNFαantibodies of the present invention, as obtained according to ExamplesXIII and XIV against a broad spectrum of in vitro TNF biologicalactivities.

EXAMPLE XVI In Vivo Activity and Efficacy of cA2 Antibody

[0372] Evidence that the potent in vitro inhibiting and/or neutralizingcapability of cA2 is manifest in vivo was obtained. Earlier animalstudies showed that administration of TNF to experimental animals mimicsthe pathology state obtained with either Gram-negative bacterialinfection or direct endotoxin administration (Tracey et al., infra(1986); Tracey et al., infra (1987); Lehmann et al. infra).

[0373] An in vivo model wherein lethal doses of human TNF areadministered to galactosamine-sensitized mice (Lehmann, V. et al.,infra) is substantially modified for testing the capability of both themurine and chimeric anti-TNFα antibodies of the present invention, asobtained according to Examples XIII and XIV above, to inhibit orneutralize TNF in vivo. An i.p. challenge with 5 μg (0.25 mg/kg) of TNFresulted in 80-90 percent mortality in untreated control animals and inanimals treated i.v. 15-30 minutes later with either saline or 2 mg/kgcontrol antibody (a chimeric IgG1 derived from murine 7E3 anti-plateletmAb). In contrast, treatment with both the murine and chimeric anti-TNFαantibodies of the present invention, as obtained according to ExamplesXIII and XIV, is expected to reduce mortality to 0-30 percent with 0.4mg/kg of antibody, and to 0-10 percent with 20 mg/kgs. These expectedresults indicate that both the murine and chimeric anti-TNFα antibodiesof the present invention, as obtained according to Examples XIII andXIV, are capable of inhibiting and/or neutralizing the biologicalactivity of TNF in vivo as well as in vitro. TABLE 4 Prevention of HumanTNF-Induced Lethality by Chimeric A2 Outcome (Survivors/Total) AntibodyExperiment #1 Experiment #2 None 1/10 N.D. Control Ab, 2 mg/kg 2/10 1/10 cA2 (2 mg/kg) (p = 0.0001) 9/10 10/10 (p = 0.0055) cA2 (0.4 mg/kg)7/10 10/10 (p = 0.0001) (p = 0.07)

EXAMPLE XVII cA2 MAb Safety in Chimpanzees

[0374] The epitope specificity of A2 can be for an epitope whichpredominates in humans and chimpanzees. Therefore, the chimpanzee waschosen as a relevant mammalian species to determine the toxicologicalpotential and provide safety information for cA2. Chimpanzees were dosedat levels of 15 mg/kg for four to five consecutive days and 30 mg/kgonce or for three consecutive days. No adverse clinical signs, and nochanges considered to be cA2 treatment related were observed in themonitored parameters including routine hematology and blood chemistry.Thus, doses of up to 30 mg/kg for three consecutive days were welltolerated in chimpanzees.

EXAMPLE XVIII Clinical Activity and Efficacy of cA2 Antibody

[0375] Chimeric IgG1 anti-human TNF MAb cA2 was administered to healthymale human volunteers as patients. One hour after receiving 4 ng/kg ofan NIH reference endotoxin, the volunteers were administered eithersaline, as a control, or 0.01, 0.10 or 10 mg/kg of cA2 in apharmaceutically acceptable form. TNF levels in serum were measured overtime and were found to show a dose dependent decrease in peak TNF levelswith no TNF being detected in volunteers receiving a 10 mg/kg dose ofcA2. Accordingly, therapy with an anti-TNF antibody of the presentinvention is expected to inhibit TNF-mediated effects in humans.

[0376] Patients receiving endotoxin developed pronounced leukopeniathought to be due to margination of white blood cells. As the whiteblood cells become activated, they can attach to endothelial receptorswith resultant endothelial damage. At doses of 1.0 to 10.0 mg/kg, thisleukopenia is prevented, whereas, at 0.01 and 0.1 mg/kg dosages, a dropin white cell count was observed. The drop was most pronounced among thepolymorph cell line. In all patients there was a subsequentleukocytosis, which was unchanged by treatment with anti-TNF antibodycA2. This blocking effect on white blood cell margination is expected torepresent a protective effect against the endothelial damage associatedwith TNF. It is expected in the art that this TNF-related endothelialdamage plays a significant role in the morbidity and mortalityassociated with sepsis, and it is therefore expected that the anti-TNFantibodies of the present invention will provide a protective effectagainst these damaging effects, as presented herein.

EXAMPLE XIX Treatment of Sepsis in Humans Using a Chimeric Anti-TNFAntibody

[0377] The chimeric anti-TNF MAb cA2 has been used in two phase P/IIstudies. In a phase I/II study in septic patients, 20 patients with thesepsis syndrome received a single dose of either 0.1, 1.0, 5.0 or 10milligrams of cA2 per kilogram bodyweight. Another 60 patients received100 milligrams of HA-1A, a human anti-lipid A Mab currently underevaluation for gram negative sepsis, followed with either placebo or1.0, 5.0, or 10 milligrams cA2 per kilogram bodyweight. The cA2 wasadministered as a single, intravenous infusion over a 60 minute period.Clinical assessment, vital signs, and laboratory parameters weremeasured before, during and periodically for 28 days after the infusion.In this study, cA2 was well tolerated. No adverse events were reportedas “probably” or “definitely” related to cA2. All deaths were reportedas “definitely not” related to cA2.

[0378] Accordingly, human treatment of rheumatoid arthritis in humanpatients was expected, and found, to provide a suitable treatment, asdescribed herein.

EXAMPLE XX Clinical Treatment of Rheumatoid Arthritis By a Anti-TNFAntibody or Peptide of the Present Invention

[0379] A Phase I open label study was conducted for methods andcompositions of the present invention using a chimeric anti-TNF MAb forthe treatment of patients with severe refractory rheumatoid arthritis.Nine patients were enrolled in the study. The first five patients weretreated with chimeric anti-TNF antibody (cA2), 10 mg/kg as a single doseinfused over a period of two hours. These patients were subsequentlyretreated with a second infusion of 10 mg/kg on day 14 of the study. Thesecond group of five patients received an infusion of 5 mg/kg on thefirst day of the study. They were then treated with additional infusionsof 5 mg/kg on days 5, 9, and 13. Four of the planned five patients inthis second group have been treated to date.

Preparation, Administration, and Storage of Test Material

[0380] The chimeric monoclonal anti-TNF antibody was supplied insingle-use glass vials containing 20 mL with 100 mg of anti-TNF (5mg/mL). The anti-TNF antibody was stored at 2-8° C. Prior to infusion,the antibody was withdrawn from the vials and filtered through alow-protein-binding 0.22 μm filter. This filtered antibody was thendiluted to a final volume of 300 mL with normal saline. The 300 mLantibody preparation was then infused via an in-line filter over aperiod of not less than two hours.

[0381] Prior to each repeat infusion of study medication a test dose of0.1 mL of the infusion was diluted in 10 mL of normal saline andadministered by slow TV push over 5 minutes. The patient was observedfor 15 minutes for signs or symptoms of an immediate hypersensitivityreaction. If no reaction was observed in this time period, the fall dosewas administered as described above.

Administration Protocol

[0382] Group 1 (patients 1-5): a total of2 infusions, on day I and day15 of the trial; dosage 10 mg/kg on each occasion;

[0383] Group 2 (patients 6-9): a total of 4 infusions, on days 1, 5, 9and 13 of the trial; dosage 5 mg/kg on each occasion.

[0384] All infusions were administered iv over 2 hours in a total volumeof cA2 +saline of 300 ml. Infusions subsequent to the first in anypatient were preceded by a small test dose administered as an iv push.All patients had at least three years of disease activity withrheumatoid arthritis. The patients ranged in age from 23 to 63. Allpatients had failed therapy with at least three different DMARD (DiseaseModifying Anti-Rheumatic Drug). Six of the nine patients had serumrheumatoid factors, and all nine patients had erosions present onX-rays.

Clinical Monitoring

[0385] Patients were monitored during and for 24 hours after infusionsfor hemodynamic change, fever or other adverse events. Clinical andlaboratory monitoring for possible adverse events was undertaken on eachfollow-up assessment day. Clinical response parameters were performed atthe time-points as specified in the flow charts present in Tables 9A and9B. These evaluations were performed prior to receiving any infusions.

[0386] Clinical response studies will be comprised of the followingparameters:

[0387] 1. Number of tenderjoints and assessment of pain/tenderness Thefollowing scoring will be used:

[0388] 0=No pain/tenderness

[0389] 1=Mild pain. The patient says it is tender upon questioning.

[0390] 2=Moderate pain. The patient says it is tender and winces.

[0391] 3=Severe pain. The patient says it is tender and winces andwithdraws.

[0392] 2. Number of swollen joints

[0393] Both tenderness and swelling will be evaluated for each jointseparately. MCP's, PIP's etc. will not be considered as one joint forthe evaluation.

[0394] 3. Duration of morning stiffness (in minutes)

[0395] 4. Grip strength

[0396] 5. Visual analog pain scale (0-10 cm)

[0397] 6. Patients and blinded evaluators will be asked to assess theclinical response to the drug. Clinical response will be assessed usinga subjective scoring system as follows:

[0398] 5=Excellent response (best possible anticipated response)

[0399] 4=Good response (less than best possible anticipated response)

[0400] 3=Fair response (definite improvement but could be better)

[0401] 2=No response (no effect)

[0402] 1=Worsening (disease worse)

[0403] Measurement of index of disease activity is scored according tothe following Table 5. TABLE 5 Clinical Characteristics of Patients 1-5Previous Disease Treatment Concomitant Patient Age/ Duration Rheumat.Erosions/ (DMARDs Anti-rheumatic Number Sex (years) Factor Nodules only)Therapy 01 48/F 7 +ve −ve/+ve *Sal, DP, Myo, **Pred 5 mg Aur, MTX, Aza,Chl 02 63/F 7 −ve +ve/−ve Sal, Myo, DP Para 1-2 g 03 59/M 3 +ve +ve/−veAur, Chl, Myo, Pred 10 mg; MTX, Sal Ind 225 mg 04 56/M 10  +ve +ve/−veMyo, DP, Aza, Pred 12.5 mg, Sal Ibu 2 g, Para 1-2 g 05 28/F 3 +ve+ve/−ve Myo, Sal, DP, Pred 8 mg, Aza Para 1-2 g, Cod 16 mg

[0404] TABLE 6 Clinical Characteristics of Patients 6-9 Previous DiseaseTreatment Concomitant Patient Age/ Duration Rheumat. Erosions/ (DMARDsAnti-rheumatic Number Sex (years) Factor Nodules only) Therapy 06 40/M 3 +ve +ve/−ve *Sal, Chl, Aur **Nap 1 g 07 54/F  7 −ve +ve/−ve DP, Myo,Sal, Para 1-2 g Aza, MTX Cod 16-32 mg 08 23/F 11 +ve +ve/−ve Chl, Myo,Sal, Pred 7.5 mg, Dicl MTX, Aza 100 mg, Para 1- 2 g, Dext 100-200 mg 0951/F 15 −ve +ve/+ve Myo, Chl, DP, Pred 7.5 mg, Dicl MTX 125 mg, Para 1-3 g

[0405] TABLE 7 Disease Activity at Entry for Patients 1-5 ESR Grip(mm/hr CRP Pain Number Ritchie Strength normal (mg/l; Morning (10—10Swollen Articular L/R (mm/ ranges: normal IDA Patient Stiffness cm onJoints Index Hg; max F <15; range (range Number (mins) VAS) (0-28)(0-69) 300) M <10) <10) 1-4) 01 60 3.9 19 30 108/107 35  5 2.67 02 202.7 25 31 67/66 18 14 2.0 03 90 4.9 14 16 230/238 48 44 2.5 04 30 6.9 1712 204/223 24 35 2.33 05 90 5.7 28 41 52/89 87 107  3.0

[0406] TABLE 8 Disease Activity at Entry for Patients 6-9 ESR Grip(mm/hr CRP Pain Number Ritchie Strength normal (mg/l; Morning (10—10Swollen Articular L/R (mm/ ranges: normal IDA Patient Stiffness cm onJoints Index Hg; max F <15; range (range Number (mins) VAS) (0-28)(0-69) 300) M <10) <10) 1-4) 06 120 5.0  3  4 260/280 23 33 2.33 07 1057.4 27 31 59/80 25 10 2.83 08 270 9.3 17 37  73/125 35 31 3.17 09 1804.5 20 26 53/75 15 33 2.5

[0407] All patients have tolerated the infusions of chimeric anti-CD4and no serious adverse reactions have been observed. Specifically, noepisodes of hemodynamic instability, fevers, or allergic reactions wereobserved in association with the infusions. Patients have notexperienced any infections.

[0408] Although this is a non-blinded study, all patients experiencedimprovement in their clinical assessments of disease status, as well inbiochemical parameters of inflammation measured in their serum.

[0409] Clinical assessments, including the duration of early morningstiffness; the assessment of pain on a visual analogue scale; totalcount of swollen joints; Ritchie articular index (a scaled score whichassesses the total number of tender joints and the degree ofjointtenderness); and Index of Disease Activity (a scaled score whichincorporates several clinical and laboratory parameters), showedimpressive improvements compared to controls. These improvements weretypically in the range of an 80% drop from the baseline score; a degreeof improvement which is well beyond the amount of improvement that canbe attributed to placebo response. In addition, the duration of theseimprovements was for six to eight weeks in most cases, a duration ofresponse far longer than would be anticipated from a placebo.

[0410] The improvements in clinical assessments were corroborated byimprovements in biochemical inflammatory parameters measured in serum.The patients showed rapid drops of serum C-reactive protein, usually inthe range of 80% from the baseline. Reductions in the erythrocytesedimentation rate, usually in the range of 40%, were also observed.Circulating soluble TNF receptors were also decreased following therapy.The durations of the biochemical responses were similar to the durationof the clinical responses.

[0411] Preliminary assessment of immune responses to the chimericanti-TNF antibody has shown no antibody response in the first fourpatients.

[0412] In summary, the preliminary evaluation of the results of thisPhase I trial indicate that treatment of patients with advancedrheumatoid arthritis with anti-TNF MAb of the present invention is welltolerated and anti-TNF treatment is associated with rapid and markedimprovement in clinical parameters of disease activity, including earlymorning stiffness, pain, and a number of tender and swollen joints; andis accompanied by improvement of biochemical parameters of inflammation.

[0413] Although this was an open label study, the magnitude of theclinical improvements is well beyond the degree of improvement thatwould be anticipated from a placebo response, such that the presentinvention is shown to have significant clinical efficacy for treatingrheumatoid arthritis. TABLE 9A Flowchart for Chimeric Anti-TNF StudyCO168TRA Group 1 (10 mg/kg at day 1 and day 14) Pre- Screen- Wk 0 Wk 0Wk Wk 2 Wk Wk Wk Wk Scr ing D 1 D 2 1 D 14 3 4 6 8 Consent X DemographyX Physical X X Examination Pregnancy X Test Weight X X X X Vital Signs XX* X X X* X X X X Anti-TNF X X Infusion Labs, see X X¹ X X X¹ X X X XChart Clinical X X X¹ X X X X (Safety) Clinical X X¹ X X¹ X X X X(Response) Synovial X X7 Biopsy Response X Evaluation Hematology + X X¹X X¹ X X X X ESR Biochemistry X X¹ X X¹ X X X X Urinalysis X¹ X X¹ X X XX CRP + RF X¹ X X¹ X X X X Serum X¹ X X¹ X X X X Cytokines PBL X X XPharmacokinetic X^(#) X^(#) X^($) HACA Response X¹ X X¹ X X X X

[0414] TABLE 9B Flowchart for Chimeric Anti-TNF Study CO168TRA Group 2(5 mg/kg at days 1, 5, 9 and 13, 4 times total) Wk Wk Wk Wk Wk Pre-Screen- 0 0 0 0 1 Wk Wk Wk Wk Wk Scr ing D 1 D 2 D 5 D 9 D 13 2 3 4 6 8Consent X Demography X Physical X X Examination Pregnancy X Test WeightX X X X X X Vital Signs X X* X X* X* X* X X X X X Anti-TNF X X X XInfusion Labs, see X X¹ X X¹ X¹ X¹ X X X X X Chart Clinical X X¹ X¹ X¹ XX X X X (Safety) Clinical X X¹ X¹ X X X X X (Response) Synovial X X7Biopsy Response X Evaluation Hematology + X X¹ X¹ X X X X X ESRBiochemistry X X¹ X¹ X X X X X Urinalysis X¹ X¹ X X X X X CRP + RF X¹ X¹X X X X X Serum Cytokines X¹ X¹ X X X X X PBL X X X X PharmacokineticX^(#) X^(#) X^($) X^($) X^($) HACA Response X¹ X¹ X X X X X

[0415] TABLE 10 Measurement of the Index of Disease Activity (DA)Variables of Disease Activity Morning Grip Ritchie IDA Stiffness PainStrength Articular Hemoglobin (g/dl) Score (mm) (VAS, cm)* (mmHg) IndexMale Female ESR 1 10 0-2.4 >200 0 >14.1 >11.7 0.20 2 10-30 2.5-4.450-200 1-7 13-14 10.8-11.6 21-45 3 31-120 4.5-6.4 30-49 8-17 10-8.4-10.7 46-80 12.9 4 >120 6.5-10 <30 >18 <9.9 <8.3 >81

[0416] Conclusions (1)

[0417] Safety of anti-TNF in RA

[0418] Anti-TNF was safe and very well tolerated:

[0419] no hemodynamic, febrile or allergic episodes;

[0420] no infections;

[0421] no clinical adverse events;

[0422] a single laboratory adverse event only, probably unrelated toanti-TNF.

[0423] Conclusions (2)

[0424] Efficacy of anti-TNF in RA

[0425] Anti-TNF therapy resulted in:

[0426] rapid and marked improvements in EMS, pain and articular index inmost patients;

[0427] slower but marked improvement in swollen joint score, maximal by3-4 weeks;

[0428] rapid and impressive falls in serum CRP, and a slower fall inESR;

[0429] normalization of CRP and ESR in some patients;

[0430] rapid falls in serum C4d (a complement breakdown product) andIL-6 in patients where these indices were elevated at entry.

[0431] Duration of clinical improvements variable, with rebound in somepatients at 6-8 weeks.

[0432] Accordingly, the present invention has been shown to haveclinical efficacy in human patients for treating TNF involvedpathologies using TNF MAbs of the present invention, such as fortreating rheumatoid arthritis. Additionally, the human clinical use ofTNF antibodies of the present invention in humans is also shown tocorrelate with in vitro data and in vivo animal data for the use ofanti-TNF antibodies of the present invention for treating TNF-relatedpathologies.

EXAMPLE XXI Treatment of Crohn's Disease in Humans Using Anti-TNFαAntibodies.

[0433] Case history SB.

[0434] This 16 year old patient has a history of Crohn's disease sinceage 12. She was suffering from diarrhoea, rectal blood loss, abdominalpain, fever and weight loss. She showed perianal lesions, severe colitisand irregularity of the terminal ileum. She was treated withprednisolone (systemic and local) and pentasa. This resulted inremission of the disease, but she experienced extensive side effects ofthe treatment. She experienced severe exacerbations at age 12 and 12yrs, 5 months, (Immuran™ added), 12 yrs, 9 months, 13 yrs, 5 months, and14 yrs, 10 months. She experienced severe side effects (growthretardation, morbus Cushing, anemia, muscle weakness, delayed puberty,not able to visit school).

[0435] At 15 yrs, 11 months, she was diagnosed with a mass in the rightlower quadrant. She had a stool frequency of 28 times per week (with asmuch as 10 times per day unproductive attempts). The Crohn's index was311, the pediatric score 77.5. The sedimentation rate was elevated.Albumen and hemoglobin reduced. Before the first treatment the score was291 and pediatric score was 60, and she would possibly have to lose hercolon. She was infused on compassionate grounds with 10 mg/kg cA2,without any side effects noticed. One week after treatment hersedimentation rate was reduced from 66 to 32 mm. The Crohn's index was163 and pediatric score 30. She was reported to feel much better and thefrequency of the stools was reduced greatly. There was apparently nomore diarrhoea, but normal faeces. On Oct. 15th, before the secondinfusion she had gained weight, had a sedimentation rate of 20 mm, analbumen of 46 h/l, Crohn's index 105, pediatric score 15. There seemedto be improvement on video endoscopy. A second infusion was performed at16 yrs.

[0436] The patient was greatly improved after the second infusion. Aendoscopy showed only 3 active ulcers and scar tissue.

[0437] This is in contrast with her colon on admission when the thoughtwas that her colon should be removed. This case history shows a dramaticimprovement of severe Crohn's disease upon treatment with cA2 anti-TNFantibody. TABLE 11 Case History SB 11y, 8m Physical Diarrhoea, rectalblood loss, abdominal Examination pain, fever (40%) weight loss perianallesions Sigmoidoscopy Severe colitis, probably M. Crohn EnterolysisIrregularity terminal ileum Therapy Prednisolone 10 mg 3 dd Pentasa 250mg 3 dd Enema (40 mg prednisone, 2 g 5 ASA) ml 1 dd Result Remission,however: extensive side effects of prednisone and stunting growth ActionPrednisone 11y, 11m Exacerbation Same clinical picture as 11y, 8mSigmoidoscopy Recurrence of colitis (grade IV) in last 60 cm and anusTherapy Prednisolone 40 mg 1 dd Pentasa 500 mg 3 dd Enema 1 dd ResultBetter 12y, 5m Severe Exacerbation Despite intensive treatmentSigmoidoscopy Extensive perianal and sigmoidal lesions; active diseaseTherapy Continued + Immuran ™ 25 mg 1 dd Result Slight improvement,however still growth retardation, cushing, anaemia, muscle weaknessAction Prednisone 12y, 9m Exacerbation Sigmoidoscopy Extensive (activecolitis, polyps) Action Prednisone: 30 mg 1 dd, Immuran ™ 50 mg 1 ddPentasa 500 mg 3 dd Enema 2 dd Result Still needs enemas with prednisoneand oral prednisone. Delayed puberty, stunting growth 14y, 10m SevereExacerbation Weight loss, abdominal pain, fever Ileoscopy Active colitis(grade IV), perianal lesions. Terminal ileum normal Result No remissionstill fever, poor appetite, weight loss, diarrhea, not able to visitschool Important Findings 14y, 11m 151.9 cm; 34 kg; t = 38° C.,Abdominal mass in right lower quadrant; stool frequency 28 per week(however goes 10-15 times a day but most often without success); ESR 55mm; Hb 6.2 mmol/1; Ht 0.29 l/l; alb. 38.4 g/l Crohn's Dis./Act Index:311 Pediatric score: 77.5 14y, 11.2m 151.8 cm; 34.6 kg (before 1stinfusion) Crohn's Dis/Act Index: 291 Pediatric score: 60 14y, 11.4m151.8 cm; 34.6 kg; ESR 332 mm; Hb 5.7 mmol/l Crohn's Dis/Act Index: 163Pediatric score: 30 15y, 0m 152.1 cm: 34.8 kg (before 2nd infusion)Feels like she has never felt before. Parents also very enthusiastic;ESR 30 mm: Hb 6.3 mol/l; Ht 0.32 11; Alb 46 g/l Crohn Dis/Act Index: 105Pediatric Score: 15 Videoendoscopy: Improvement No problems or sideeffects observed during and following infusion.

[0438] Accordingly, anti-TNF antibodies according to the presentinvention, as exemplified by cA2, are shown to provide successfultreatment of TNF related pathologies, as exemplified by Crohn's disease,in human patients with no or little side effects.

EXAMPLE XXII Treatment of Arthritis in Humans Using ChimericInmunoglobulin Chain of the Present Invention Patient Selection

[0439] Twenty patients were recruited, each of whom fulfilled therevised American Rheumatism Association criteria for the diagnosis of RA(Arnett et al., Arthritis Rheum. 31:315-324 (1988). The clinicalcharacteristics of the patients are shown in Table 12. The study groupcomprised 15 females and 5 males, with a median age of 51 years (range23-72), a median disease duration of 10.5 years (range 3-20) and ahistory of failed therapy with standard disease—modifying anti-rheumaticdrugs (DMARDs; median number of failed DMARDs: 4, range 2-7). Seventeenwere seropositive at entry or had been seropositive at some stage oftheir disease, all had erosions on X-Rays of hands or feet, and 3 hadrheumatoid nodules. All patients had active disease at trial entry, asdefined by an Index of Disease Activity (IDA; Mallya et al., Rheumatol.Rehab. 20:14-17 (198 1) of at least 1.75, together with at least 3swollen joints, and were classed as anatomical and functional activitystage 2 or 3′ (Steinbrocker et al., JAMA 140:659-662 (1949). The pooleddata for each of the clinical and laboratory indices of disease activityat the time of screening for the trial (up to 4 weeks prior to trialentry), and on the day of trial entry itself (week 0), are shown inTables 13 and 14. TABLE 12 Demographic Features of 20 Patients withRefractory Rheumatoid Arthritis Pat Age/Sex DD (yr) Previous DMARDsConcomitant therapy 1 48/F 7 SSZ, DP, GST, AUR, MTX, Pred 5 mg AZA, HCQ2 63/F 7 SSZ, GST, DP Para 1-2 g 3 59/M 3 AUR, HCQ, GST, Pred 10 mg;Indo 225 mg MTX, SSZ 4 56/M 10 GST, DP, AZA, SSZ Pred 12.5 mg; Ibu 2 g;Para 1-2 g 5 28/F 3 GST, SSZ, DP, AZA Pred 8 mg; Para 1-2 g; Cod 16 mg 640/M 3 SSZ, HCQ, AUR Nap 1 g 7 54/F 7 DP, GST, SSZ, Para 1-2 g; Cod16-32 mg AZA, MTX 8 23/F 11 HCQ, GST, SSZ, Pred 7.5 mg; Dicl 100 mg;Para 1-2 g; Dex MTX, AZA 100-200 mg 9 51/F 15 GST, HCQ, DP, MTX Pred 7.5mg; Dicl 125 mg; Para 1-3 g 10 47/F 12 SSZ, CYC, MTX Ben 4 g 11 34/F 10DP, SSZ, MTX Pred 10 mg; Para 1.5 g; Cod 30-90 mg 12 57/F 12 GST, MTX,DP, AUR Asp 1.2 g 13 51/F 7 SSZ, AZA Para 1-4 g 14 72/M 11 GST, DP, AZA,MTX Pred 5 mg; Para 1-4 g; Cod 16-63 mg 15 51/F 17 HCQ, DP, SSZ, MTX Asp0.3 g 16 62/F 16 GST, DP, SSZ, Para 1-4 g; Cod 16-64 mg MTX, AZA 17 56/F11 SSZ, DP, GST, Pred 7.5 mg; Eto 600 mg; Para 1-2 g; Dext MTX, HCQ, AZA100-200 mg 18 48/F 14 GST, MTX, DP, SS, Pred 7.5 mg; Indo 100 mg; Para1-3 g ZAUR, AZA 19 42/F 3 SSZ, MTX Fen 450 mg; Ben 6 g; Cod 30 mg 2047/M 20 GST, DP, SSZ, AZA Pred 10 mg; Para 1-3 g # phosphate; Nap =naprosyn; Dicl = diclofenac; Dext = dextropropoxyphene; Ben =benorylate; Asp = aspirin; eto = etodolac; Fen = fenbufen.

[0440] TABLE 13 Changes in Clinical Assessments Following Treatment ofRheumatoid Arthritis Patients with cA2 Patient Grip Grip Assess- PainSwollen Strength Strength ment Morning Score Ritchie Joints (L) (R)(grades Week of Stiffness (0-10) Index (0-28) (0-300) (0-300) IDA (1-improved Trial (min) cm (0-69) number mm Hg mm Hg 4) 0-3) Screen 135 7.423 16 84 96 3 NA 0-600) (4-9.7) (4-51) (4-28) (45-300) (57-300) (2.3-3.3) p value 0 180 7.1 28 18 77 92 2 NA (20-600) (2.7-9.7) (4-52) (3-27)(52-295) (50-293) (2-3.5) p value 1 20 2.6 13(2-28) 13.5 122 133 2 1(0-180) (0.6-7.8) <0.001; (1-25) (66-300) (57-300) (1.5- (1-3) <0.001<0.001 <0.002 >0.05 >0.05 >0.05 3.3) NA <0.001 p value 2 15 3.0 13 11.5139 143 2 1.5 (0-150) (0.2-6.4) (1-28) (1-22) (75-300) (59-300) (1.5-(1-3) <0.001 <0.001 <0.001 <0.003; <0.03; >0.05 3.2) NA <0.02 >0.05<0.001 p value 3 5 2.2 8 6(1-19) 113 142 2 2 (0-150) (0.2-7.4) (0-22)<0.001; (51-300) (65-300) (1.2- (1-2) <0.001 <0.001 <0.001<0.002 >0.05 >0.05 3.2) NA <0.001 p value 4 15 1.90 10 6(0-21) 124 1481.8 2 (0-90) (0.1-5.6) (0-17) <0.001; (79-300) (64-300) (1.3- (1-2)<0.001 <0.001 <0.001 <0.002 <0.02; <0.03; 2.7) NA >0.05 >0.05 <0.001 pvalue 6 5 1.9 6 5 119 153 1.7 2 (0-90) (0.1-6.2) (0-18) (1-14) (68-300)(62-300) (1.3- (1-2) <0.001 <0.001 <0.001 <0.001 <0.04; <0.05; 2.8)NA >0.05 >0.05 <0.001 p value 8 15 2.1 8 7 117 167 1.8 2 (0-60)(0-2-7.7) (1-28) (1-18) (69-300) (52-300) (1.5- (1-3) <0.001 <0.001<0.001 <0.001 <0.03; <0.03; 2.8) NA >0.05 >0.05 <0.001

[0441] TABLE 14 Changes in Laboratory Measures Following Treatment ofRheumatoid Arthritis patients with cA2 Platelet RF Week of Hgb WBC ×Count × ESR CRP SAA Inverse Trial g/liter 10/liter 10/liter mm/hourmg/liter mg/ml titer Screen 117 7.9 353 59 42 ND ND (98-146) (3.9-15.2)(274-631) (18-87) (9-107) P value 0 113 9.0 341 55 39.5 245 2,560(16(97-144) (4.9-15.7) (228-710) (15-94) (5-107) (18-1900) 0-10,240) pvalue 1 114 8.5 351 26 5 58 ND (96-145) (3.6-13.6) (223-589) (13-100)(0-50) (0-330) >0.05 >0.05 >0.05 >0.05 <0.001 <0.001; <0.003 p value 2112 8.2 296 27 5.5 89 ND (95-144) (4.3-12.7) (158-535) (10-90) (0-80)(11-900) >0.05 >0.05 <0.04; <0.02; <0.001; <0.02; >0.05 >0.05 <0.003<0.04 p value 3 110 9.0 289 27 7 ND ND (89-151) (3.7-14.4) (190-546)(12-86) (0-78) >0.05 >0.05 <0.03; <0.04; <0,01; >0.05 >0.05 <0.002 pvalue 4 112 8.2 314 23 10 ND ND (91-148) (4.7-13.9) (186-565) (10-87)(0-91) >0.05 >0.05 >0.05 <0.04; <0.004; >0.05 <0.02 p value 6 116 9.1339 23 8 ND ND (91-159) (2.9-13.9) (207-589) (12-78)(0-59) >0.05 >0.05 >0.05 <0.03; <0.001 >0.05 p value 8 114 7.6 339 30 6ND 480 (40- (94-153) (4.2-13.5) (210-591) (7-73) (0-65)05,120) >0.05 >0.05 >0.05 >0.05 <0.001 >0.05

[0442] All DMARDs were discontinued at least 1 month prior to trialentry. Patients were allowed to continue on a nonsteroidalanti-inflammatory drug and/or prednisolone (<12.5 mg/day) during thetrial. The dosage of these agents was kept stable for 1 month prior totrial entry and during the course of the trial, and no parenteralcorticosteroids were allowed during these periods. Simple analgesicswere allowed ad libitum. Patients with other serious medical conditionswere excluded. Specific exclusions included serum creatinine >150umol/liter (normal range 60-120 umol/liter), hemoglobin (Hgb) <90gm/liter (normal range 120-160 gm/liter [females]; 135-175 gm/liter[males]), white blood cell count (WBC) <4×10 g/liter (normal range4−11×10⁹/liter), platelet count <100×10 g/liter (normal range150-400xlO⁹/liter), and abnormal liver fimction tests or activepathology on chest X-Ray.

[0443] All patients gave their informed consent for the trial, andapproval was granted by the local ethics committee.

Treatment

[0444] The cA2 antibody was stored at 4° C. in 20 ml vials containing 5mg of cA2 per milliliter of 0.01 M phosphate buffered saline in 0.15Msodium chloride at a pH of 7.2 and was filtered through a 0.2 μm sterilefilter before use. The appropriate amount of cA2 was then diluted to atotal volume of 300 ml in sterile saline and administered intravenouslyvia a 0.2 μm in-line filter over a 2 hour period.

[0445] Patients were admitted to hospital for 8-24 hours for eachtreatment, and were mobile except during infusions. The trial was of anopen, uncontrolled design, with a comparison of two treatment schedules.Patients 1 to 5 and 11 to 20 received a total of 2 infusions, each of 10mg/kg cA2, at entry to the study (week 0) and 14 days later (week 2).Patients 6 to 10 received 4 infusions of 5mg/kg activity includedcomplete blood counts, C-reactive protein (CRP; by rate nephelometry)and the erythrocyte sedimentation rate (ESR; Westergren). Follow-upassessments were made at monthly intervals after the conclusion of theformal trial period, in order to assess the duration of response.

[0446] Analysis of improvement in individual patients was made using twoseparate indices. Firstly, an index of disease activity (IDA) wascalculated for each time point according to the method of Mallya andMace (Mallya et al., Rheumatol. Rehab. 20:14-17 (1981), with inputvariable of morning stiffness, pain score, Ritchie Index, grip strength,ESR and Hgb. The second index calculated was that of Paulus (Paulus etal., Arthritis Rheum. 33:477-484 (1990) which uses input variables ofmorning stiffness, ESR, joint pain/tenderness, joint swelling, patient'sand physician's global assessment of disease severity. In order tocalculate the presence or otherwise of a response according to thisindex, two approximations were made to accommodate our data. The 28swollen joint count used by us (nongraded; validated in Fuchs et al.,Arthritis Rheum. 32:531-537 (1989)) was used in place of the moreextensive graded count used by Paulus, and the patient's and physician'sglobal assessments of response recorded by us were approximated to theglobal assessments of disease activity used by Paulus infra. In additionto determining response according to these published indices, weselected 6 disease activity assessments of interest (morning stiffness,pain score, Ritchie index, swollen joint count, ESR and CRP) andcalculated their mean percentage improvement. We have used FIGS. 24 and25 to give an indication of the degree of improvement seen in respondingpatients.

Immunological Investigations

[0447] Rheumatoid factors were measured using the rheumatoid arthritisparticle agglutination assay (PAPA, FujiBerio Inc., Tokyo, Japan), inwhich titers of {fraction (1/160)} or greater were consideredsignificant. Rheumatoid factor isotypes were measured by ELISA(Cambridge Life Sciences, Ely, UK). The addition of cA2 atconcentrations of up to 200 μg/ml to these assay cA2, at entry, and days4, 8 and 12. The total dose received by the 2 patient groups wastherefore the same at 20 mg/kg.

Assessment Safety Monitoring

[0448] Vital signs were recorded every 15 to 30 minutes duringinfusions, and at intervals for up to 24 hours post infusion. Patientswere questioned concerning possible adverse events before each infusionand at weeks 1, 2, 3, 4, 6, and 8 of the trial. A complete physicalexamination was performed at screening and week 8. In addition, patientswere monitored by standard laboratory tests including complete bloodcount, C3 and C4 components of complement, IgG, IgM and IgA, serumelectrolytes, creatinine, urea, alkaline phosphatase, aspartatetransaminase and total bilirubin. Sample times for these tests werebetween 0800 and 0900 hours (pre-infusion) and 1200-1400 hours (24 hourspost completion of the infusion). Blood tests subsequent to day 1 wereperformed in the morning, usually between 0700 and 1200 hours. Urineanalysis and culture were also performed at each assessment point.

Response Assessment

[0449] The patients were assessed for response to cA2 at weeks 1, 2, 3,4, 6 and 8 of the trial. The assessments were all made between 0700 and1300 hours by the same observer. The following clinical assessments weremade: duration of morning stiffness (minutes), pain score (0 to 10 cm ona visual analog scale), Ritchie Articular Index (maximum 69; Ritchie etal., Quart. J. Med. 147:393-406 (1968)), number of swollen joints (28joint count; validated in Fuchs et al., Arthritis Rheum. 32:531-537(1989), grip strength (0 to 300 mm Hg, mean of 3 measurements per handby sphygmomanometer cuff) and an assessment of function (the StanfordHealth Assessment Questionnaire (HAQ) modified for British patients;34). In addition, the patients' global assessments of response wererecorded on a 5-point scale (worse, no response, fair response, goodresponse, excellent response). Routine laboratory indicators of diseasesystems did not alter assay results (data not shown). Antinuclearantibodies were detected by immunofluorescence on HEpo 2 cells(Biodiagnostics, Upton, Worcs. UK) and antibodies to extractable nuclearantigens were measured by counter immunoelectrophoresis withpoly-antigen extract (Biodiagnostics). Sera positive byimmunofluorescence were also screened for antibodies to DNA by the Farrassay (Kodak Diagnostics, Amersham, UK). Anti-cardiolipin antibodieswere measured by ELISA (Shield Diagnostics, Dundee, Scotland). Serumamyloid A (SAA) was measured by sandwich ELISA (Biosource International,Camarillo, Calif., USA). Antiglobulin responses to the infused chimericantibody were measured by an in-house ELISA, using cA2 as a capturereagent.

Cytokine Assays

[0450] Bioactive TNF was measured in sera using the WEHI 164 clone 13cytotoxicity assay (Espevik et al., J. Imm. Methods 95:99-105 (1986).Total IL-6 was measured in sera using a commercial immunoassay (MedgenixDiagnostics, SA, Belgium) and by a sandwich ELISA developed “in house”using monoclonal antibodies provided by Dr. F. di Padova (Basel,Switzerland). Microtiter plates were coated with monoclonal antibody LNI314-14 at a concentration of 3 ug/ml for 18 hours at 4° C. and blockedwith 3% bovine serum albumin in 0.1M phosphate buffered saline, pH 7.2.Undiluted sera or standards (recombinant hIL 6, 0-8.1 ug/ml) were addedto the wells in duplicate and incubated for 18 hours at 4° C. Bound IL-6was detected by incubation with monoclonal antibody LNI 110-14 for 90minutes at 37° C., followed by biotin labeled goat anti-murine IgG2b for90 minutes at 37° C. (Southern Biotechnology, Birmingham, Ala.). Theassay was developed using streptavidin—alkaline phosphatase (SouthernBiotechnology) and p-nitrophenylphosphate as a substrate and the opticaldensity read at 405 nm.

Statistics

[0451] Comparisons between week 0 and subsequent time points were madefor each assessment using the Mann - Whitney test. For comparison ofrheumatoid factor (RAPA) titers, the data were expressed as dilutionsbefore applying the test.

[0452] This was an exploratory study, in which prejudgements about theoptimal times for assessment were not possible. Although it has not beencommon practice to adjust for multiple statistical comparisons in suchstudies, a conservative statistical approach would require adjustment ofp values to take into account analysis at several time points. The pvalues have therefore been presented in two forms: unadjusted, and aftermaking allowance for analysis at multiple time points by use of theBonferroni adjustment. Where p values remained <0.001 after adjustment,a single value only is given. A p value of <0.05 is consideredsignificant.

Results Safety of cA2

[0453] The administration of cA2 was exceptionally well tolerated, withno headache, fever, hemodynamic disturbance, allergy or other acutemanifestation. No serious adverse events were recorded during the 8-weektrial. Two minor infective episodes were recorded, patient 15 presentedat week 2 with clinical features of bronchitis and growth of normalcomnmensals only on sputum culture. She had a history of smoking and ofa similar illness 3 years previously. The illness responded promptly totreatment with amoxicillin, but her second cA2 infusion was withheld andthe data for this patient are therefore not analyzed beyond week 2.Patient 18 showed significant bacteriuria on routine culture at week 6(>10⁵/ml; lactose fermenting coliform), but was asymptomatic. Thiscondition also responded promptly to amoxicillin.

[0454] Routine analysis of blood samples showed no consistent adversechanges in hematological parameters, renal function, liver function,levels of C3 or C4 or immunoglobulins during the 8 weeks of the trial.Four minor, isolated and potentially adverse laboratory disturbanceswere recorded. Patient 2 experienced a transient rise in blood urea,from 5.7 mmol/liter to 9.2 mmol/liter (normal range 2.5 to 7mmol/liter), with no change in serum creatinine. This change wasassociated with the temporary use of a diuretic, prescribed for anon-rheumatological disorder. The abnormality normalized within 1 weekand was classified as “probably not” related to cA2. Patient 6experienced a transient fall in the peripheral blood lymphocyte count,from 1.6 to 0.8×10⁹/liter (normal range 1.0-4.8×10⁹/liter). Thisabnormality normalized by the next sample point (2 weeks later), was notassociated with any clinical manifestations and was classified as“possible related” to cA2. Patients 10 and 18 developed elevated titersof anti-DNA antibodies at weeks 6 and 8 of the trial, with elevatedanti-cardiolipin antibodies being detected in patient 10 only. Bothpatients had a pre-existing positive antinuclear antibody and patient 10had a history of borderline lymphocytopenia and high serum IgM. Therewere no clinical features of systemic lupus erythematosus and thelaboratory changes were judged possibly related to cA2.

Efficacy of cA2 Disease Activity

[0455] The pattern of response for each of the clinical assessments ofdisease activity and the derived IDA are shown in Table 13. All clinicalassessments showed improvement following treatment with cA2, withmaximal responses from week 3. Morning stiffness fell from a median of180 minutes at entry to 5 minutes at week 6 (p<0.001, adjusted),representing an improvement of 73%. Similarly, the Ritchie Indeximproved from 28 to 6 at week 6, (p<0.001, adjusted, 79% improvement)and the swollen joint count fell from 18 to 5, (p<0.001, adjusted, 72%improvement). The individual swollen joint counts for all time pointsare shown in FIG. 24. Grip strength also improved; the median gripstrength rose from 77 (left) and 92 (right) mm Hg at entry to 119 (left)and 153 (right) mmHg at week 6 (p<0.04, p<0.05, left and rightrespectively; p>0.05 after adjustment for multiple comparisons). The IDAshowed a fall from a median of 3 at entry to 1.7 at week 6 (p<0.001,adjusted). Patients were asked to grade their responses to cA2 on a 5point scale. No patient recorded a response of “worse” or “no change” atany point in the trial. “Fair”, “good” and “excellent” responses wereclassed as improvements of 1, 2 and 3 grades respectively. At week 6,the study group showed a median of 2 grades of improvement (Table 13).

[0456] We also measured changes in the patients' functional capacity,using the HAQ modified for British patients (range 0-3). The median(range) HAQ score improved from 2(0.9-3) at entry to 1.1 (0-2.6) by week6, (p<0.00l;p<0.002 adjusted).

[0457] The changes in the laboratory tests which reflect diseaseactivity are shown in Table 14. The most rapid and impressive changeswere seen in serum CRP, which fell from a median of 39.5 mg/liter atentry to 8 mg/liter by week 6 of the trial (p<0.001, adjusted; normalrange <10 mg/liter), representing an improvement of 80%. Of the 19patients with elevated CRP at entry, 17 showed falls to the normal rangeat some point during the trial. The improvement in CRP was maintained inmost patients for the assessment period (Table 14 and FIG. 25); theexceptions with high values at 4 and 6 weeks tended to be those with thehighest starting values (data not shown). The ESR also showedimprovement, with a fall from 55 mm/hour at entry to 23 mm/hour at week6 (p<0.03; p>0.05 adjusted; 58% improvement; normal range <10 mm/hour,<15 mm/hour, males and females respectively). SAA levels were elevatedin all patients at trial entry, and fell from a median of 245 mg/ml to58 mg/ml at week 1 (p<0.003, adjusted; 76% improvement; normal range<10/mg/ml) and to 80 mg/ml at week 2 (p<0.04, adjusted). No significantchanges were seen in Hgb, WBC or platelet count at week 6, although thelatter did improve at weeks 2 and 3 compared with trial entry (Table14).

[0458] The response data have also been analyzed for each individualpatient. The majority of patients had their best overall responses atweek 6, at which time 13 assessed their responses as “good” while 6assessed their responses as “fair”. Eighteen of the 19 patients whocompleted the treatment schedule achieved an improvement in the index ofDisease Activity (Mallya et al., Rheumatol. Rehab. 20:14-17 (1981) of0.5 or greater at week 6, and 10 achieved an improvement of 1.0 orgreater. All patients achieved a response at week 6 according to theindex of Paulus (Paulus et al., Arthritis Rheum. 33:477-484 (1990).Finally, all patients showed a mean improvement at week 6 in the 6selected measures of disease activity (as presented above) of 30% orgreater, with 18 of the 19 patients showing a mean improvement of 50% orgreater.

[0459] Although the study was primarily designed to assess theshort-term effects of cA2 treatment, follow-up clinical and laboratorydata are available for those patients followed for sufficient time(number=12). The duration of response in these patients, defined as theduration of a 30% (or greater) mean improvement in the 6 selecteddisease activity measures, was variable, ranging from 8 to 25 (median14) weeks.

[0460] Comparison of the clinical and laboratory data for patientstreated with 2 infusions of cA2 (each at 10 m/kg) compared with thosetreated with 4 infusions (each at 5 mg/kg) showed no significantdifferences in the rapidity or extent of response (data not shown).

Immunological Investigations and Cytokines

[0461] Measurement of rheumatoid factor by RAPA showed 14 patients withsignificant titers (>{fraction (1/160)}) at trial entry. Of these, 6patients showed a fall of at least 2 titers on treatment with cA2, whilethe remaining patients showed a change of 1 titer or less. No patientshowed a significant increase in RF titer during the trial. The medianRF titer in the 11 patients fell from ½, 560 at entry to {fraction(1/480)} by week 8 (p>0.05; Table 14). Specific RF isotypes weremeasured by ELISA, and showed falls in the 6 patients whose RAPA haddeclined significantly, as well as in some other patients. Median valuesfor the three RF isotypes in the 14 patients seropositive at trial entrywere 119, 102 and 62 IU/ml (IgM, IgG and IgA isotypes respectively) andat week 8 were 81, 64 and 46 IU/ml (p>0.05).

[0462] We tested sera from the first 9 patients for the presence ofbioactive TNF, using the WEHI 164 clone 13 cytotoxicity assay (Espeviket al.,J. Imm. Methods 95:99-105 (1986). In 8 patients, serum setsspanning the entire trial period were tested, while for patient 9, onepre-trial, one period were tested, patient, one pre, one intermediateand the last available sample only were tested. The levels of bioactiveTNF were below the limit of sensitivity of the assay in the presence ofhuman serum (1 pg/ml). Since production of CRP and SAA are thought to beregulated in large part by IL-6, we also measured serum levels of thiscytokine, using 2 different assays which measure total IL-6. In theMedgenix assay, IL-6 was significantly elevated in 17 of the 20 patientsat entry. In this group, levels fell from 60 (18-500) pg/ml to 40(0-230) pg/ml at week 1 (p>0.05; normal range <10 pg/ml) and to 32(0-210) pg/ml at week 2 (p<0.005, p<0.01, adjusted). These results weresupported by measurement of serum JL-6 in the first 16 patients in aseparate ELISA developed in-house. IL-6 was detectable in 11 of the 16,with median (range) levels falling from 210 (25-900) pg/ml at entry to32 (01,700) pg/ml at week 1 (p<0.02, p<0.04, adjusted; normal range <10pg/ml) and to 44 (0-240) pg/ml at week 2 (p<0.02, p<0.03, adjusted).

[0463] We tested sera from the first 10 patients for the presence ofanti-globulin responses to the infused chimeric antibody, but none weredetected. In many patients however, cA2 was still detectable in serumsamples taken at week 8 and this can have interfered with the ELISA.

Discussion

[0464] This is the first report describing the use of anti-TNFαantibodies in human autoimmune disease. Many cytokines are produced inrheumatoid synovium, but we chose to target specifically TNFα because ofmounting evidence that it was a major molecular regulator in RA. Thestudy results presented here support that view and allow three importantconclusions to be drawn.

[0465] First, treatment with cA2 was safe and the infusion procedure waswell tolerated. Although fever, headache, chills and hemodynamicdisturbance have all been reported following treatment with anti CD4 oranti CDw52 in RA, such features were absent in our patients. Alsonotable was the absence of any allergic event despite repeated treatmentwith the chimeric antibody, although the interval between initial andrepeat infuisions can have been too short to allow maximal expression ofany anti-globulin response. The continuing presence of circulating cA2at the conclusion of the trial may have precluded detection ofantiglobulin responses, but also implied that any such responses werelikely to be of low titre and/or affinity. Although we recorded 2infective episodes amongst the study group, these were minor and theclinical courses were unremarkable. TNFα has been implicated in thecontrol of listeria and other infections in mice (Havell et al.,J.Immunol. 143:2894- 2899 (1989), but our limited experience does notsuggest an increased risk of infections after TNFα blockade in man.

[0466] The second conclusion concerns the clinical efficacy of cA2. Thepatients we treated had long-standing, erosive, and for the most partseropositive disease, and had each failed therapy with several standardDMARDs. Despite this, the major clinical assessments of disease activityand outcome (morning stiffness, pain score, Ritchie index, swollen jointcount and HAQ score) showed statistically significant improvement, evenafter adjustment for multiple comparisons. All patients graded theirresponse as at least “fair”, with the majority grading it as “good”. Inaddition, all achieved a response according to the criteria of Paulusand showed a mean improvement of at least 30% in 6 selected diseaseactivity measures.

[0467] The improvements in clinical assessments following treatment withcA2 appear to be at least as good as those reported following treatmentof similar patients with antileukocyte antibodies. The two therapeuticapproaches can already be distinguished, however, by their effects onthe acute phase response, since in several studies of antileukocyteantibodies, no consistent improvements in CRP or ESR were seen. Incontrast, treatment with cA2 resulted in significant falls in serum CRPand SAA, with normalization of values in many patients. The changes wererapid and marked, and in the case of CRP, sustained for the duration ofthe study (Table 14). The falls in ESR were less marked, achievingstatistical significance only when unadjusted (Table 14).

[0468] These results are consistent with current concepts that implicateTNFα in the regulation of hepatic acute phase protein synthesis, eitherdirectly, or by control of other, secondary, cytokines such as IL-6(Fong et al.,J. Exp. Med. 170:1627-1633 (1989); Guerne et al.,J. Clin.Invest. 83:585-592 (1989)). In order to investigate the mechanism ofcontrol of the acute phase response in our patients, we measured serumTNFα and IL-6 before and after cA2 treatment. Bioactive TNFα was notdetectable in baseline or subsequent sera. We used 2 different assaysfor IL-6, in view of previous reports of variability between differentimmunoassays in the measurement of cytokines in biological fluids(Roux-Lombard et al., Clin. Exp. Rheum. 10:515-520 (1992), and bothdemonstrated significant falls in serum IL-6 by week 2. These findingssupport the other objective laboratory changes induced by cA2, andprovide in vivo evidence that TNFα is a regulatory cytokine for IL-6 inthis disease. Amongst the other laboratory tests performed, rheumatoidfactors fell significantly in 6 patients.

[0469] Neutralization of TNFα can have a number of beneficialconsequences, including a reduction in the local release of cytokinessuch as IL-6 and other inflammatory mediators and modulation of synovialendothelial/leukocyte interactions. cA2 can also bind directly tosynovial inflammatory cells expressing membrane TNFoC, with subsequentin situ cell lysis.

[0470] The results obtained in this small series have importantimplications, both scientifically and clinically. At the scientificlevel, the ability of the neutralizing antibody, cA2, to reduce acutephase protein synthesis, reduce the production of other cytokines suchas IL-6, and significantly improve the clinical state demonstrates thatit is possible to interfere with the cytokine network in a useful mannerwithout untoward effects. Due to the many functions and overlappingeffects of cytokines such as IL-1 and TNFα, and the fact that cytokinesinduce the production of other cytokines and of themselves, there hadbeen some pessimism as to whether targeting a single cytokine in vivowould have any beneficial effect (Kingsley et al., Immunol. Today12:177-179 (1991), Trentham, Curr. Opin. Rheumatol. 3:369-372 (1991)).This view is clearly refuted. On the clinical side, the results ofshort-term treatment with cA2 are significant and confirm that TNFC isuseful as a new therapeutic target in RA.

EXAMPLE XXI Treatment with Chimeric Anti-TNF in a Patient with SevereUlcerative Colitis.

[0471] The patient is a 41 year old woman with long term ulcerativecolitis, which was diagnosed by endoscopy and histology. She has apancolitis, but the main disease activity was left-sided. There were noextra-intestinal complications in the past. Maintenance therapyconsisted of Asacol™. Only one severe flair-up occurred 4 yearspreviously and was successfully treated with steroids.

[0472] At beginning month one, she was admitted elsewhere because of avery severe flair-up of the ulcerative colitis. Treatment consisted ofhigh doses of steroids intravenously, antibiotics, asacol and TotalParental Nutrition. Her clinical condition worsened and a colectomy wasconsidered.

[0473] At end of month one, she was admitted at the internal ward of theAMC. Her main complaints consisted of abdominal pains, frequent waterstools with blood and mucopus and malaise.

[0474] Medication: ASACOL 2 dd 500 mg, orally

[0475] Di-Adresone-T 1 dd 100--mg, intravenously

[0476] Flagyl 3 dd 500 mg, intravenously

[0477] Fortum 3 dd 1 gram, intravenously

[0478] Total parental nutrition via central venous catheter

[0479] On physical examination the patient was moderately ill with aweight of 55 kg and a temperature of 36° C. Jugular venous pressure wasnot elevated. Blood pressure was 110/70 mm Hg with a pulse rate of 80per minute. No lymphadenopathy was found. Oropharynx was normal. Centralvenous catheter was inserted in situ with no signs of inflammation atthe place of insertion. Normal auscultation of the lungs and heart. Theabdomen was slightly distended and tender. Bowel sounds where reduced.Liver and spleen where not enlarged. No signs of peritonitis. Rectalexamination was normal.

[0480] All cultures of the stools where negative.

[0481] Plain x-ray of the abdomen; slightly dilated colon. Nothumb-printing, no free air, no toxic megacolon.

[0482] Sigmoidoscopy; (video-taped) Very severe inflammation with deepulcers. Dilated rectum and sigmoid. Because of danger of perforation thecolor, the endoscopy was limited to the racto-sigmoid. No biopsies wheretaken.

[0483] Conclusion at time of admission: Severe steroid resistantflair-up of ulcerative colitis.

[0484] Antibiotics were stopped, because no improvement was noticed andthere was no temperature.

[0485] After informed consent of the patient, treatment was started with10 mg/kg bodyweight (a 550 mg) of cA2 chimeric monoclonal anti-TNF(Centocor) given intravenously over 2 hours (according the protocol ofcA2 used in severe Crohn's disease).

[0486] During the infusion there were no complaints. Vital signs weremonitored and were all normal. Before and after infusion blood sampleswere drawn. Two days after infusion she had less abdominal pain, thestool frequency decreased and no blood was seen in the stools any more.However she developed high temperature (40° C.). Blood cultures werepositive for Staphylococcus epidermidis. Infection of the central venouscatheter was suspected. The catheter was removed and the sameStaphylococcus was cultured from the tip of the central venous catheter.During this period she was treated with antibiotics for three days.After this her temperature dropped and she recovered substantially.Steroids were tapered off to 40 mg of prednisone daily.

[0487] After 14 days sigmoidoscopy was repeated and showed a remarkableimprovement of the mucosa with signs of reepithelization. There were nosigns of bleeding, less mucopus and even some normal vascular structureswere seen.

[0488] At four months she was discharged.

[0489] At the outpatient clinic further monitoring was done weekly.Patient is still improving. Stool frequency is two times per day withoutblood or mucopus. Her laboratory improved, but there is still anaemia,probably due to iron deficiency. A colonoscopy is planned in the nearbyfuture.

[0490] Our conclusion is that this patient had a very severe flair-up ofher ulcerative colitis. She was refractory to treatment and a totalcolectomy was seriously considered. After infusion of cA2 the clinicalcourse improved dramatically in spite of the fact that there was acomplication of a sepsis which was caused by the central venouscatheter.

EXAMPLE XXIV Treatment of Rheumatoid Arthritis in Humans with cA2Antibody Patients

[0491] Patients were recruited from the clinics of four cooperatingtrial centers or after referral from outside physicians. Patients aged18-75 were included if they met the criteria of the American College ofRheumatology for the diagnosis of rheumatoid arthritis, had had diseasefor at least six months, had a history of failed treatment with at leastone disease modifying anti-rheumatic drug (DMARD) and had evidence oferosive disease on radiography of hands and feet. In addition, patientshad to have active disease, as defined by the presence of six or moreswollen joints plus at least three of four secondary criteria (durationof morning stiffness ≧45 minutes; ≧6 tender or painful joints;erythrocyte sedimentation rate (ESR) ≧28 mm/h; C-reactive protein (CRF)≧20 mg/L). Patients with severe physical incapacity (Steinbrocker classIV) or with clinically evident joint ankylosis were excluded. Otherexclusion criteria included: severe anaemia (haemoglobin <8.5 g/dL);leucopenia (white cells <3.5×10⁹/L, neutrophils <1.5×10⁹/L) orthrombocytopenia (100×10⁹/L); elevation of liver function tests to overthree times the upper limit of normal or of serum creatine to over 150μmol/L; or active pathology on chest film. Patients were also excludedif they had a history of previous administration of murine monoclonalantibodies, a history of cancer or HIV infection, or current otherserious medical conditions. Female patients of child-bearing age had tobe using an effective method of birth control and to have a negativepregnancy test before entry.

[0492] No patient had received other experimental drugs targeted to TNF(e.g., oxpentifylline) in the previous three months. Patients takingdisease-modifying anti-rheumatic drugs at screening were withdrawn fromtheir therapy at least four weeks before entry. Patients taking low-doseoral corticosteroids (prednisolone ≦12.5 mg per day) or non-steroidalanti-inflammatory drugs at screening were allowed to continue on stabledoses. Additional steroids by injection or other routes were notallowed. Simple analgesics were freely allowed.

[0493] All patients gave their informed consent for the trial, which wasapproved by each of the local regional ethics committees.

Study Infusions

[0494] The cA2 antibody was supplied as a sterile solution containing 5mg cA2 per ml of 0.01 mol/L phosphate-buffered saline in 0.15 mol/Lsodium chloride with 0.01% polysorbate 80, pH 7.2. The placebo vialscontained 0.1% human serum albumin in the same buffer. Before use, theappropriate amount of cA2 or placebo was diluted to 300 mL in sterilesaline by the pharmacist, and administered intravenously via a 0.2 μmin-line filter over 2 hours. The characteristics of the placebo and cA2infusion bags were identical, and the investigators and patients did notknow which infusion was being administered.

Assessments

[0495] Patients were seen at an initial screening visit and if eligible,were entered within four weeks. On the day of entry, patients wereadmitted to the hospital and randomized (in blocks of 6, stratified forcenter) to one of three groups (24 per group). The first group receiveda single infusion of placebo. The other two groups received one infusionof cA2, 1 mg/kg (“low dose”) and 10 mg/kg (“high dose”). The doses ofcA2 were chosen on the basis of experience in the open-label trial andby extrapolation from the anti-TNF-treated collagen-arthritis mice.

[0496] Patients were monitored for adverse events during infusions andregularly thereafter, by interviews, physical examination, andlaboratory testing.

[0497] Before the start of the trial, all clinical observers agreed on astandard technique to assess joints, and to establish protocols for thecollection of other clinical data. In each center, patients wereassessed by the same clinical observer at each evaluation visit, usuallybetween 0800 and 1100 hour. Clinical observers were additionally blindedto the results of laboratory testing for acute-phase measures (ESR andCRP).

[0498] The six primary disease-activity assessments were chosen to allowanalysis of the response in individual patients according to the Paulusindex. The assessments contributing to this index were the tender andswollen joint scores (60 and 58 joints, respectively, hips not assessedfor swelling; graded 0-3), the duration of morning stiffniess (minutes),the patient's and observer's assessment of disease severity (on a 5point scale, ranging from 1 (symptom-free) to 5 (very severe) and ESR.Patients were considered to have responded if at least four of the sixvariables improved, defined as at least 20% improvement in thecontinuous variables, and at least two grades of improvement orimprovement from grade 1 to 1 in the two disease-severity assessments(Paulus 20% response). Improvements of at least 50% in the continuousvariables were also used (Paulus 50%).

[0499] Other disease-activity assessments included the pain score (0-10cm on a visual analogue scale (VAS)), an assessment of fatigue (0-10 cmVAS), and grip strength (0-300 mm Hg, mean of three measurements perhand by sphygmomanometer cuff).

[0500] The ESR was measured at each study site with a standard method(Westergen). CRP (Abbott fluorescent polarizing immunoassay) andrheumatoid factor (rheumatoid-arthritis particle-agglutination assay(RAPA, FujiBerio, Tokyo); tiftes 2160 were taken to be important) weremeasured in stored frozen serum samples shipped to a central laboratory.

Statistics

[0501] The analysis was on the basis of intention to treat. The samplesize was chosen as having an 80% probability of achieving astatistically significant (p<0.05) result if the true response rateswere 10% and 40% in the placebo and 10 mg/kg cA2 groups, respectively.Fisher's exact test was used to compare the groups for baseline sexratio and rheumatoid factor status and for Paulus response rates.Comparisons between groups for other demographic features and forindividual disease activity assessments were by analysis of variance, orCochran-Mantel-Haenszel statistics where appropriate (baselinecomparison of disease-modifying anti-rheumatic drugs usage, patient'sand observer's assessments of disease severity/activity). The Paulus 20%response rate at week 4 was defined as the primary efficacy endpoint,with other time points and variables considered supportive. Levels ofsignificance were therefore not adjusted for multiple comparisons.

Results

[0502] Seventy-two patients were initially randomized. One patientpresented two weeks after treatment with 1 mg/kg cA2 with probablepneumonia that required admission to the hospital. The patient waswithdrawn and, according to protocol, another patient was recruited.Thus, the intention-to-treat analysis brought the number analyzed in the1 mg/kg group to 25 patients and the total number to 73.

[0503] The three groups were well-matched at entry, with no significantdifferences in age, sex ratio, disease duration, number of faileddisease-modifying anti-rheumatic drugs, or percentage of patients withsignificant titre of rheumatoid factor (Table 15). Demographic data weresimilar between the four sites. The patients had active disease atentry, as judged by the presence of multiple tender and swollen joints,high pain scores, substantial morning stiffness, raised acute-phasemeasures (Table 16). Comparison between groups revealed no significantdifferences for any of the clinical and laboratory indices of diseaseactivity at entry.

[0504] The response rates at Paulus 20% and 50% are shown in Table 17.Only 2 of 24 placebo recipients achieved a 20% response at week 4. Bycontrast, 19 of 24 patients treated with 10 mg/kg cA2 achieved aresponse by week 4 (pO.0001 compared with placebo). The response ratesin the 1 mg/kg group were intermediate, with 11 of 25 patientsresponding at week 4 (p=0.0083). Analysis of the Paulus 50% responseshowed similar differences between the groups, with 14 of 24 high-dosecA2 patients responding (p=0.0005), compared with 2 of 24 patients inthe placebo group. Analysis of the response data for corticosteroid useshowed that patients who were taking steroids behaved no differently intheir responses from non-steroid-treated patients.

[0505] Although secondary to the differences in overall response rates,analysis of changes in individual disease-activity assessments was alsoof interest (Table 16). Significant improvements were seen in both cA2groups for each of the clinical assessments. For many assessments,maximum mean improvements in cA2-treated groups exceeded 60%. Among thelaboratory measures, significant falls were seen in both cA2 groups forESR, CRP, and platelet counts, with the best improvements seen in thehigh-dose group. The changes in CRP were particularly rapid in onset andimpressive in extent, with many individual patients achieving normalconcentrations (10 mg/L, data not shown). In addition, significantimprovements relative to placebo were seen for haemoglobin, especiallyin the high-dose cA2 group. Trends towards a fall in white cell count(from increased counts at entry) in both cA2 groups supported thechanges in other laboratory measures, but did not reach statisticalsignificance (Table 16).

[0506] Detailed time response profiles for six disease-activityassessments common to the American College of Rheumatology and theEuropean League Against Rheumatism core-sets showed rapid and highlysignificant falls in the cA2-treated groups compared with placebo, withsignificant inter-group differences evident as early as 24 and 72 h (CRPand all other assessments, respectively).

[0507] Seeking possible dose-response relations, we compared responserates between the cA2 groups. We found no difference in 20% or 50%Paulus responses at week 2, but significantly higher response rates forthe high-dose group at week 4 (likelihood ratio 1.8, 95% CI 1.1, 2.9,p=0.0186; 2.1, 1.1, 4.1, p=0.0450, for Paulus 20% and 50%,respectively). A similar analysis for each of the individualdisease-activity assessments showed no greater benefit with the higherdose at week 2 of the study, except for haemoglobin (least squares meandifference 0.5, 95% CI 0.1, 0.9, p=0.021). By week 4, however, somediminution of the response in the 1 mg/kg group was evident for severalassessments; responses in the 120 mg/kg group were maintained (Table16). As a result, significantly better responses were seen at this timein the high-dose group, including pain score (least-squares meandifference −1.8, 95% CI −3.4 −0.2, p=0.036), right (28.4, 5.4, 51.3,p=0.018) and left grip strength (20.6, 3.3, 37.9, p=0.022), observer'sassessment of disease severity (−0.8, −1.3, −0.4, p<0.035), ESR (−15.0,−23.6 to −1.4 p=0.035), CRP (−20.7, −32.1, −9.2, p<0.001), andhaemoglobin (0.5, 0.0, 1.0, p=0.042).

[0508] The infusions of cA2 and placebo were well tolerated, with noepisodes of fever or hemodynamic disturbance. The adverse eventsrecorded during the 4 weeks after treatment are shown in Table 18. Inall, two-thirds of the adverse events occurred in the cA2 groups.Infections formed the largest group, with 5 infections recorded in the 1mg/kg group and 1 each in those receiving 10 mg/kg cA2 and placebo.

[0509] Of the 72 initially randomized, 2 patients had severe adverseevents. One was the patient with probable pneumonia. The patientrecovered fully with treatment, but was withdrawn and replaced. Thisevent was judged “possibly” related to cA2. A second patient presented 1week after treatment with 10 mg/kg cA2 with a pathological fracture ofthe clavicle, but continued in the study. In retrospect, a minor bonyabnormality was evident on an X-ray film taken pretreatment, and theevent was judged “probably not” related to cA2. TABLE 15 DemographicFeatures Group 1 mg/kg cA2 10 mg/kg cA2 Placebo (n = 24) (n = 25) (n =24) Age (yr) 48-1 (11-9) 56-2 (12-2) 50-6 (13-1) M/F 7/17 5/25 4/20Disease Duration 9-0 (7-3) 7-5 (4-8) 7-3 (5-2) Previous Drugs* 3-7 (1-9)2-8 (1-5) 3-1 (1-7) Rheumatoid Factor 7% 96% 75% (seropositive)

[0510] TABLE 16 Disease Activity Assessments Data Summary Statisticalanalysis vs. Placebo 1 mg/kg 10 mg/kg 1 mg/kg cA2 10 mg/kg cA2 Assess WkPlacebo cA2 cA2 Least-sq 95% CI p Least-sq 95% CI p Tender joint count 027.8 (13.5) 29.1 (14.1) 28.1 (12.7) 2 25.7 (16.6) 12.1 (10.2) 11.1 (6.9)−14.8 −21.2, −8.4 <0.001 −14.8 −20.2, −9.5 <0.001 4 26.2 (15.5) 16.9(12.1) 11.3 (9.8) −10.9 −16.4, −5.3 <0.001 −15.2 −21.2, −9.2 <0.001Swollen Joint count (0-58) 0 23.4 (10.5) 21.4 (10.6) 21.8 (11.5) 2 24.2(12.1) 11.1 (8.1) 8.2 (5.5) −10.9 −15.6, −6.3 <0.001 −14.4 −19.6, −9.2<0.001 4 23.0 (11.2) 12.9 (8.8) 8.6 (6.4) −8.2 −12.8, −3.6 0.001 −12.7−17.8, −7.5 <0.001 Pain Score (0-10 cm) 0 6.8 (1.8) 6.6 (2.6) 6.7 (2.5)2 6.9 (2.6) 2.5 (2.6) 2.6 (2.1) −1.3 −5.7, −2.9 <0.001 −4.3 −5.8, −2.8<0.001 4 6.9 (2.5) 4.2 (2.9) 2.5 (1.8) −2.6 −4.2, −0.9 0.003 −4.3 −5.8,−2.9 <0.001 Morning Stiffness (min) 0 132.3 (286.7) 142.0 143.1 (106.5)2 150.6 (284.0) (122.0) 10.3 (14.9) −88.9 −147.5, −30.3 0.004 −101.2−156.4, −16.1 <0.001 4 172.3 (300.1) 27.4 (48.7) 8.3 (13.6) −33.4−156.4, 89-6 0.592 −124.8 −188.9, −60.8 <0.001 99.6 (286.3) FatigueScore (0-19 cm) 0 6.3 (2.3) 6.5 (2.6) 5.6 (2.4) 2 5.8 (2.9) 3.2 (2.7)2.8 (2.3) −2.6 −4.3, −1.0 −0.003 −2.3 −3.9, −0.7 0.006 4 5.6 (3.0) 3.8(2.8) 2.3 (1.7) −1.9 −3.6, −0.2 0.028 −2.6 4.3, −1.0 0.003 GripStrength, right (0-300 mm Hg) 0 120.7 (50.2) 102.4 (48.8) 117.0 (64.1) 2122.7 (51.5) 161.8 (78.3) 175.3 (79.1) 55.8 32.6, 79.0 <0.001 56.4 35.0,77.3 <0.001 4 119.1 (50.2) 131.8 (65.0) 175.2 (78.6) 31.3 15.6, 46.9<0.001 59.9 35.9, 83.9 <0.001 Grip Strength, left (0-300 mm Hg) 0 120.0(58.4) 100.8 (46.8) 108.4 (50.5) 2 123.3 (64.9 152.4 (72.0) 157.2 (65.1)46.7 23.7, 69.6 <0.001 45.4 28.9, 52.0 <0.001 4 120.9 (58.4) 126.6(65.8) 155.1 (60.9) 25.0 6.5, 43.5 0.010 45.8 28.9, 62.7 <0.001 DiseaseSeverity, patient (1-5) 0 3.8 (0.5) 3.7 (0.5) 3.6 (0.6) 2 3.8 (0.8) 3.5(0.7) 2.6 (1.0) −1.2 −1.6, −0.8 <0.001 −1.1 −1.6 −0.6 <0.001 4 3.3 (0.8)3.0 (0.8) 2.6 (0.8) −0.7 −1.2, −0.3 0.002 −1.2 −1.7 −0.8 <0.001 DiseaseSeverity, Observer 0 3.7 (0.7) 3.7 (0.5) 3.6 (0.7) 2 3.5 (0.8) 2.5 (0.8)2.3 (0.6) −1.0 −1.5, −0.6 <0.001 −1.2 −1.6, −0.8 <0.001 4 3.6 (2.0) 3.0(1.0) 2.2 (0.6) −0.6 −1.1, −0.1 0.036 −1.4 −1.9, −1.0 <0.001 ESR (mm/h)0 63.1 (24.8) 58.1 (25.5) 63.1 (27.6) 2 67.0 (27.4) 41.8 (24.6) 42.4(25.2) −23.1 −35.9, −10.4 <0.001 −24.9 −39.0, −10.9 <0.001 4 65.1 (29.8)52.4 (32.3) 42.7 (24.6) −10.7 −26.4, 5.1 0.185 −22.5 −38.7, −6.3 0.009CRP (mg/L) 0 64 (42) 67 (41) 64 (36) 2 53 (30) 39 (39) 28 (29) −19.1−34.1, −4.1 0.016 −24.3 −38.8, −9.8 0.002 4 60 (42) 58 (39) 35 (29) −7.7−20.5, 5.1 0.239 −28.8 −33.7, −12.9 <0.001 Haemoglobin g/dL 0 11.6 (1.6)11.8 (1.3) 11.0 (1.1) 2 10.9 (1.5) 11.5 (1.2) 11.2 (1.1) 0.4 0.0, 0.70.052 0.8 0.5, 1.2 <0.001 4 10.9 (1.7) 11.7 (1.2) 11.4 (1.2) 0.6 0.1,1.0 0.022 1.1 0.6, 1.5 <0.001 WBC (×10⁹/L) 0 10.7 (3.5) 10.1 (3.5) 9.0(2.1) 2 10.5 (2.9) 9.2 (3.2) 8.4 (2.5) −0.8 −1.9, 0.4 0.202 −0.4 −1.4,0.6 0.414 4 10.3 (3.2) 9.3 (4.3) 7.7 (2.0) −0.4 −1.6, 0.8 0.500 −0.9−1.9, 0.2 0.096 Platelets (×10⁹/L) 0 447 (126) 421 (132) 400 (127) 2 471(135 375 (111) 368 (117) −69 −108, −30 0.001 −56 −89, −22 0.002 4 462(115) 406 (131) 345 (120) −29 −073, 16 0.208 −69 −103, −36 <0.001

[0511] TABLE 17 Responses According to Paulus 20% and 50% Criteria atEach Evaluation Point Data Summary 10 1 mg/kg mg/kg Statistical Analysisvs. Placebo Placebo cA2 cA2 1 mg/kg cA2 10 mg/kg cA2 n = 24 n = 25 n =24 LR 95% CI p LR 95% CI p Paulus 20% Day 3 2 (8%)  8 (32%)  7 (29%) 3.81.1, 14.0 0.0738 3.5 0.9, 13.4  0.1365 Wk 1 2 (8%) 13 (52%) 16 (67%) 6.22.1, 18.6 0.0015 8.0 3.0, 21.5  0.0001 Wk 2 3 (13%) 15 (60%) 18 (75%)5.0 2.1, 12.2 0.0008 6.0 2.7, 13.5 <0.0001 Wk 3 4 (17%) 12 (48%) 21(88%) 2.9 1.2, 7.1 0.0322 5.3 2.7, 10.3 <0.0001 Wk 4 2 (8%) 11 (44%) 19(79%) 5.3 1.7, 16.9 0.0083 9.5 3.9, 23.4 <0.0001 Paulus 50% Day 3 1 (4%) 6 (24%)  2 (9%) 5.8 1.0, 33.1 0.0983 2.0 0.2, 20.0  1.000 Wk 1 1 (4) 11(44%) 12 (50%) 10.6 2.5, 44.6 0.0019 12.0 3.0, 47.5  0.007 Wk 2 0 11(44%) 12 (50%) NA NA 0.0002 NA NA <0.0001 Wk 3 2 (8%)  7 (28%) 13 (54%)3.4 0.9, 13.0 0.1383 6.5 2.2, 19.2  0.0013 Wk 4 2 (8%)  7 (28%) 14 (58%)3.4 0.9, 13.0 0.1383 7.0 2.5, 20.0  0.0005

[0512] TABLE 18 All Adverse Events Recorded During 4 Weeks After Entry 1mg/kg 10 mg/ System Event Placebo cA2 kg cA2 Respiratory URTI 1(0) 2(0)1(1) Probable pneumonia — 1(1) — Pleuritis — 1(0) — Gastro intestinalNausea 2(0) — — Diarrhoea 1(1) — — Abdominal Pain — 2(0) — Peptic Ulcer— 1(0) — Blood loss per rectum — 1(0) — Cardiovascular Hypertension 1(0)1(1) 1(1) Peripheral oedema — 1(0) 1(0) Skin Rash 3(1) 1(0) — Infection— 2(2) — Injection site reaction — 1(1) — Neurological Dizziness 3(1) —— Headache — — 1(0) Musculoskeletal Rheumatoid nodules — 1(0) —Popliteal cyst — 1(0) — Fracture — — 1(0) Other Malaise — 1(0) — Rigors— 1(1) — Facial oedema — 1(0) — Scleritis/conjunctivitis — 1(0) —Vasculitis 1(0) — —

EXAMPLE XXV Treatment of Crohn's Disease with cA2 Antibody

[0513] This patient is a 25 year old female known with Crohn's diseasewith an eight year history, who has had several exacerbations of Crohn'sdisease. Following the birth of a child the patient developed again anexacerbation. Prednisone treatment was increased to 30 mg about 15months earlier. It was not possible to sufficiently taper offprednisone, and azathioprine was added to the therapy six months priorto antibody treatment. Remission could not be achieved, and in the endthe patient was enrolled in this trial.

[0514] At enrollment her Crohn's medication had been stable for fourmonths and consisted of mesalazine 3×250 mg, prednisone 20 mg, andazathioprine 100 mg. Her main symptoms were frequent diarrhoea,abdominal cramps and poor general well being. Her CDAI was 216, CRP 14and ESR 32. Endoscopy prior to treatment showed severe inflammation withpseudopolyps and deep ulcerations of the ascending and transverse colon.

[0515] The patient was infused with 840 mg of cA2 (10 mg/kg). From day 5and onwards through week 8 there was considerable improvement of hersymptoms, as is also reflected by a marked decrease in the CDAI. For thefirst time in many years the patient had formed stools again. CRP andESR also decreased a little, although not as markedly as the CDAI, butthese were not that much elevated prior to the treatment. Thisimprovement is also objectivated by the endoscopic findings, which showa greatly improved image at week 4 and a complete remission at week 8.

[0516] Prior to the infusion of cA2, the patient already had on herbuttocks skin eruptions which were identified as pyoderma gangrenosum.These skin lesions also improved substantially during treatment,although they have not vanished completely. Before the treatment withcA2, the patient already had problems with decreased vision but she didnot report this. During the follow up after three weeks she reportedthis and she was immediately sent to the ophthalmologist who concludedthat the reduced vision was a result of cataract due to prednisone. Thiswas the rationale for tapering off prednisone to 15 mg at week 6.

[0517] Colon biopsies taken during the 8 week endoscopy showed a mild tomoderate focal epithelial dysplasia in one of the biopsy specimens. Thisis a common finding in patients with chronic inflammatory bowel disease.However, differentiation between epithelial dysplasia associated withinflammatory bowel disease or a fragment of tubular adenoma could not bemade.

Pre-treatment Endoscopy

[0518] Smooth introduction of the scope till the bottom of the cecum.Because of slight edema it is not possible to enter the terminal ileum.Also the patient indicates that these attempts are quite painful. Whileretracting the scope the colon is inspected. Especially the promixalpart till approximately the flexural lienalis is severely inflamed andhas pseudopolyps and deep ulcerations. Also the mucosa is edematous.More distally the mucosa looks more normal. At about 15 cm, there is asmall lesion, which could be the exit of a fistula. Biopsies are takenand the entire endoscopy is taken on video. Conclusion: Unchanged imagecompared to previous endoscopy.

Four Week Endoscopy

[0519] Smooth introduction of the scope till in the terminal ileum.While withdrawing, the terminal ileum and cecum are inspected. Theinflammation is considerably less than compared to the endoscopy of 4weeks ago. There are still a few pseudopolyps and left and right a smallulceration, but compared to the endoscopy of 4 weeks ago, there is adramatic improvement of the endoscopic image. Distally the mucosa looksnormal. A diligent search has been made for the fistula opening whichwas seen last time at 15 cm of the anal ring. At this location we do seea scar with a slightly indurated edge, but at this moment it does notappear to be a real fistula opening. Conclusion: Greatly improvedendoscopic image, compared to the endoscopy of 4 weeks ago.

Eight Week Endoscopy

[0520] There are only pseudopolyps left. Conclusion: Complete remission.

EXAMPLE XXVI p55 Fusion Protein Structure

[0521] The extracellular domains of the p55 and p75 receptors wereexpressed as Ig fusion proteins from DNA constructs designed to closelymimic the structure of naturally occurring, rearranged Ig genes. Thus,the fused genes included the promoter and leader peptide coding sequenceof a highly expressed chimeric mouse-human antibody (cM-T412, Looney etal., Hum. Antibody Hybridomas 3:191-200 (1992)) on the 5′ side of theTNF receptor insert, and codons for eight amino acids of human Jsequence and a genomic fragment encoding all three constant domains ofhuman IgG1 on the 3′ side of the receptor insert position (FIGS. 27 and29).

[0522] Minor changes were introduced at the N-terminal ends of the heavychain fusion proteins so that the first two amino acids would beidentical or similar to the first two amino acids (Gln-Ile) encoded bythe cM-T412 antibody gene (from which the leader peptide originated).This was done to increase the likelihood that any interactions betweenthe N-terminal end of the mature protein and the leader peptide wouldstill result in efficient transport into the lumen of the endoplasmicreticulum. Boyd et al., Cell 62: 1031-1033 (1990). Therefore, the Asp¹and Ser² residues of naturally occurring p55 were replaced with a Ghnresidue, and the Leu¹ residue of p75 was preceded by a Gln residue inall p75 constructs. No amino acid changes were introduced at theN-terminal end of the p55 light chain fusion.

Expression vectors

[0523] PCR methodology was used to engineer cloned genes.Oligonucleotides were purchased from National Biosciences (Plymouth,Minn.). PCR amplification kits were from Perkin Elmer (Calif.) and DNAsequencing kits from U.S. Biochemical Corporation (Cleveland, Ohio).Alkaline phosphatase-conjugated goat anti-human IgG was purchased fromJackson InmunoResearch (West Grove, Pa.). ¹²⁵I-labeled human TNF wasobtained from Du Pont Company, NEN (Boston, Mass.) and unlabeledrecombinant human TNF from R&D Systems (Minneapolis, Minn). ProteinA-Sepharose beads was purchased from PHARMACIA (Piscataway, N.J.).

[0524] PCR methodology was used to engineer two cloned genes encodingthe heavy chain or light chain of an efficiently expressed murineantibody, cM-T412 (see Looney et al.), for the purpose of directing theexpression of foreign genes in a mammalian cell system. The approacheswere to effectively delete the coding region of the antibody variableregion and to place a unique restriction site in its place (Stul for theheavy chain vector and EcoRV for the light chain vector).

[0525] The resulting vector contained 2.5 kb of 5′ flanking genomic DNA,the promoter, the leader peptide coding sequence (including the leaderintron), a Stul cloning site to introduce inserts, coding sequence foreight amino acids of human J sequence Gly Thr Leu Val Thr Val Ser Ser(SEQ ID NO:6) followed by genomic sequences for the human IgGi constantregion. An analogous vector was made from the cM-T412 light chain geneexcept that an EcoRV cloning site was introduced at the carboxylterminal end of the light chain leader peptide and a different human Jsequence was encoded by the vector Gly Thr Lys Leu Glu Ile Lys (SEQ IDNO:7). Both vectors are based on plasmid pSV2-gpt and subsequent vectorderivatives that contain genomic sequences for either the heavy chain orlight chain constant regions. See Mulligan et al., Science 209:1422-1427(1980). The E. coli gpt gene allows selection of transfected cells withmycophenolic acid.

Heavy chain vector

[0526] A previously cloned EcoRI fragment containing the cM-T412 heavychain gene (Goeddel et al., Cold Spring Harbor Symp. Quant. Biol. 51:597-609(1986)) was subdloned into pUC19. This recombinant plasmid wasused as a template for two PCR reactions. In one reaction, an oligocorresponding to the “reverse” primer of the pUC plasmids and the 3′oligo 5′-CCTGGATACCTGTGAAAAGA-3′ (SEQ ID NO:8) (with half of a Stulsite; oligo was phosphorylated prior to the PCR reaction) were used toamplify a fragment containing 3kb of 5′ flanking DNA, the promoter,transcription start site and leader peptide coding sequence (includingthe leader intron). In the second reaction, the 5′ oligo 5°CCTGGTACCTTAGTCACCGTCT CCTCA-3′ (SEQ ID NO:9) (with half of a Stul site;oligo phosphorylated prior to the PCR reaction) and an oligocorresponding to the “forward” primer of pUC plasmids amplified afragment encoding eight amino acids of human J sequence Gly Thr Leu ValThr Val Ser Ser (SEQ ID NO:6) and a splice donor to allow splicing tothe human constant region coding sequence provided in another vector.The two PCR fragments were digested with EcoRI and then simultaneouslyligated into EcoRI-digested pUC19 to make pHC684 (FIG. 27).

[0527] Because the Stul site formed at the junction of the two PCRfragments was followed by a “GG” dinucleotide sequence, a dcmmethylation site was formed preventing Stul from digesting that sitewhen the DNA was grown in HB 101 strain of E coli. Therefore, theplasmid DNA was introduced into dcm-JM110 E. coli cells and reisolated.Stul was then able to cut at the junction but a second Stul site in the5′ flanking DNA was a apparent (DNA sequencing showed that Stul site toalso be followed by a GG dinucleotide and therefore also methylated). Tomake the Stul cloning site at the junction be unique, a 790 bp Xbalfragment that included only one of the two Stul sites was subdloned intopUC19 to make the vector pHC707 (FIG. 27) which was then grown in JM110cells. The Stul cloning site formed at the junction of the two PCRfragments second and third nucleotides (i.e., “CA”) of the last codon(Ala) of the signal sequence in order to maintain the appropriatetranslation reading frame (FIG. 27).

[0528] A PCR fragment encoding a protein of interest can then be ligatedinto the unique Stul site of pHC707. The insert can include atranslation stop codon that would result in expression of a “non-fusion”protein. Alternatively, a fusion protein could be expressed by theabsence of a translation stop codon, thus allowing translation toproceed through additional coding sequences positioned downstream of theStul cloning site. pH 730 contains coding sequences for all threeconstant domains of human IgG1 and was designed to accomodate the Xbalfragments of pHC707 at a unique Xbal site upstream of the IgG1 codingsequences (FIG. 28). Coding sequences in the Stul site of pHC707 wouldnot be fused directly to the IgG1 coding sequences in pHC730 but wouldbe separated by an intron sequence that partially originates from pHC707and partially from pHC730. These intron sequences would be deleted inthe cell following transcription resulting in an RNA molecule that istranslated into a chimeric protein with the protein of interest fuseddirectly to the IgG1 constant domains.

[0529] The plasmid pHC730 was a modified form of an IgG1 expression,pSV2gpt-hCyl vector described previously (Goeddel et al., Cold SpringHarbor Symp. Quant. Biol. 51:597-609 (1986)) (FIG. 28). Themodifications were (1) removal of the unique Sal1 and Xbal sitesupstream of the constant region coding sequence, (2) insertion of a Sal1linker into the unique BainHI site to allow use of Sal1 to linearize theplasmid prior to transfections, and (3) ligation into the unique EcoRIsite the cloned cM-T412 EcoRI fragment but with the Xbal fragmentflanking the V gene deleted (FIG. 28). The resulting expression vectorhad a unique Xbal site for inserting the Xbal fragments from pHC707.

Light chain vector

[0530] A previously cloned Hindlfl fragment containing the cM-T412 lightchain gene (Goeddel et al., Cold Spring Harbor Symp. Quant. Biol.51:597-609 (1986)) was subdloned into pUC19 and the resulting plasmidused as template for PCR reactions. In one PCR reaction the “reverse”pUC primer and the 3′ oligo 5′-AATAGATATCTCCTTCAACACCTGCAA-3′ (SEQ IDNO:10) (with an EcoRV site) were used to amplify a 2.8 kb fragmentcontaining 5′ flanking DNA, the promoter, transcription start site andleader peptide coding sequence (including the leader intron) of thecloned light chain gene. This fragment was then digested with HindIIIand EcoRV. In a second PCR reaction, the 5′ oligo 5′-ATCGGG ACAAAGTTGGAAATA-3′ (SEQ ID NO: 11) (with half of an EcoRV site) and the “forward”pUC primer were used to amplify a fragment encoding seven amino acids ofhuman J sequence (Gly Thr Lys Leu Glu Ile Lys) and an intron splicedonor sequence. This fragment was digested with HindIII and ligatedalong with the other PCR fragment into pUC cut with HindIII. Theresulting plasmid, pLC671 (FIG. 29), has a unique EcoRV cloning site atthe junction of the two PCR fragments with the EcoRV site positionedsuch that the first three nucleotides of the EcoRV site encoded thefirst amino acid of the mature protein (Asp).

[0531] The pLC671 HindIII insert was designed to be positioned upstreamof coding sequences for the human kappa light chain constant regionpresent in a previously described expression vector, pSV2gpt-hCk (FIG.30). However, pSV2gpt-hCk contained an EcoRV site in its gpt gene.Because it was desired that the EcoRV site in the pLC671 HindIIIfragment be a unique cloning site after transferring the fragment intopSV2gpt-hCk, the EcoRV site in pSV2gpt-hCk was first destroyed by PCRmutagenesis. Advantage was taken of the uniqueness of this EcoRV site inpSV2gpt-hCk and a Kpn1 site 260 bp upstream of the EcoRV site.Therefore, the 260 bp Kpn1-EcoRV fragment was removed from pSV2gpt-hCkand replaced with a PCR fragment that has identical DNA sequence to the260 bp fragment except for a single nucleotide change that destroys theEcoRV site. The nucleotide change that was chosen was a T to a C in thethird position of the EcoRV recognition sequence (i.e., GATATC changedto GACATC). Because the translation reading frame is such that GAT is acodon and because both GAT and GAG codons encode an Asp residue, thenucleotide change does not change the amino acid ended at that position.Specifically, pSV2gpt-hCk was used as template in a PCR reaction usingthe 5′ oligo 5′GGCGGTCT GGTACCGG-3′(SEQ ID NO:12) (with a Kpn1 site) andthe 3′ oligo 5′GTCAACAACATAGTCATCA-3′ (SEQ ID NO: 13) (with thecomplement of the Asp codon). The 260 bp PCR fragment was treated withthe Klenow fragment of DNA polymerase to fill-in the DNA ends completelyand then digested with Kpn1 . The fragment was ligated into pSV2gpt-hCkthat had its Kpn1 -EcoRV fragment removed to make pLC327 (FIG. 30).

[0532] The HindIII fragment of pLC671 was cloned into the unique HindIIIsite of pLC327 to make the light chain expression vector, pLC690 (FIG.30). This plasmid can be introduced into cells without furthermodifications to encode a truncated human kappa light chain, JCk, thatcontains the first two amino acids of the cM-T412 light chain gene,seven amino acids of human J sequences, and the light chain constantregion. Alternatively, coding sequence of interest can be introducedinto the unique EcoRV site of pLC690 to make a light chain fusionprotein.

TNF Receptor DNA Constructs

[0533] For the p55 heavy chain fusion, amino acids 3-159 of the p55extracellular domain were encoded in a PCR fragment generated using the5′ oligo 5′CACAGGTGTGTCCCCAAGGAAAA-3′ (SEQ ID NO:14) (with the Val³codon) and the 3′ oligo 5′-AATCTGGGGTAGGCACAA-3′ (SEQ ID NO:15) (withthe complement of the Ile¹⁵⁹ codon). For the p55 light chain fusion,amino acids 2-159 were encoded in a PCR fragment made using the 5′ oligo5,-AGTGTGTGTCCCCAAGG3′ (SEQ ID NO: 16) (with the Ser² codon) and thesame 3′ oligo shown above. The light chain vector contained the codonfor Asp¹ of p55. The DNA template for these PCR reactions was apreviously reported human p55 cDNA clone. (Gray et al., Proc. Natl.Acad. Sci. USA 87:7380-7384 (1990)).

[0534] A truncated light chain that lacked a variable region wasexpressed by transfecting cells with the light chain vector with noinsert in the EcoRV cloning site. The resulting protein, termed JC_(K),consisted of the first two amino acids of the cM-T412 light chain gene,seven amino acids of human J sequence (Gly Thr Lys Leu Gluhle Lys) (SEQID NO:7), and the human light chain constant region.

[0535] A non-fusion form of p55 (p55-nf) was expressed in CHO-K1 cellsusing the CMV-major immediate early promoter after introducing atranslation stop codon immediately after Ile¹⁵⁹. Secreted p55 waspurified by affinity chromatography on a TNFα column.

Transfections and ELISA Assays

[0536] All plasmids were linearized with a restriction enzyme prior tointroducing them into cells. Cells of the myeloma cell line X63-Ag8.653were transfected with 12 μg of DNA by electroporation. Cell supernatantswere assayed for IgG domains. Briefly, supernatants were incubated inplates coated with anti-human IgG Fc and then bound protein detectedusing alkaline phosphatase-conjugated anti-human and light chains.

Purification of Fusion Proteins

[0537] Cell supernatants were clarified by centrifugation followed bypassage through a 0.45 micron filter. Supernatants were adjusted to 20mM Tris-HCl, pH 8.3, 150 mM NaCl, and 1 mM EDTA (1× protein A buffer)and passed over a column of protein A-Sepharose beads. The column waswashed in 1× protein A buffer followed by 100 mM Na Citrate, pH 5.0 toelute bound bovine IgG originating from the cell media. Bound fusionprotein was then eluted in 100 mM Na Citrate, pH 3.5, neutralized with0.2 volumes 1 M Tris, and dialyzed against PBS.

TNF Cytotoxicity Assays

[0538] TNF-sensitive WEHI-164 cells (Espevik et al.,J. Immunol. Methods95:99-105 (1986)) were plated in 1 μg/ml actinomycin D at 50,000 cellsper well in 96-well microtiter plates for 3-4 hours. Cells were exposedto 40 pM TNFα or TNFβ and varying concentrations of fusion protein. Themixture was incubated overnight at 37° C. Cell viability was determinedby adding 3-[4,5-dimethyl- thiazol-2-yl]-2, 5diphenyltetrazolium bromidedye (MTT) to a final concentration of 0.5 mg/ml, incubating for 4 hoursat 37° C., lysing the cells in 0.1 N HCl, 0.1% SDS and measuring theoptical density at 550 nm wavelength.

Saturation binding analyses

[0539] Fusion proteins were captured while at a concentration of 10ng/ml in 96-well microtiter plates coated with goat anti-human Fcantibodies. Varying concentrations of ¹²⁵I-TNF (34.8 μCi/μg) were addedin PBS/1% BSA and allowed to bind for two hours at room temperature.Plates were washed and bound cpm determined. Non-specific binding wasdetermined using an irrelevant antibody.

[0540] Several different versions of the p55 fusion proteins wereexpressed. Unlike what was reported for CD4 (Capon et al., Nature337:525-531 (1989)) and IL-2 (Landolfi, J. Biol. Chem. 146:915-919(1991)) fusion proteins that also included the CHI domain of the heavychain, inclusion of a light chain proved to be necessary to getsecretion of the Ig heavy chain fusion proteins from the murine myelomacells. The light chain variable region was deleted to enable the TNF Rdomain on the heavy chain to bind TNF without steric hindrance from thelight chain.

[0541] The “double fusion” (df) protein, p55-df2, has p55 fused to boththe heavy chain and light chain and is therefore tetravalent with regardto p55. p55-sf3 has the p55 receptor (and the same eight amino acids ofhuman J sequence present in p55-sf2 and p55-df2) linked to the hingeregion, i.e., the C_(H)1 domain of the constant region is deleted.

[0542] After one or two rounds of subdloning, spent cell supernatantfrom the various cell lines were yielding 20 μg/ml (for p55-sf2) offusion protein. The proteins were purified from the spent supernatant byprotein A column chromatography and analyzed by SDS-PAGE with or withouta reducing agent. Each fusion protein was clearly dimeric in that theirM_(r) estimates from their migration through a non-reducing gel wasapproximately double the estimated M_(r) from a reducing gel. However,two bands were seen for p55-sf2 (lane 1) and p55-df2. Two lines ofevidence indicated that, in each case, the lower bands did not include alight chain while the upper bands did include a light chain. First, whenp55-sf2 containing both bands were passed over an anti-kappa column, theupper band bound to the column (lane 3) while the lower band passedthrough the column. Second, Western blots have shown that only the upperbands were reactive with anti-kappa antibodies.

[0543] It is believed that the versions of these fusion proteins that donot have a light chain (k) were not secreted to a significant degree butrather were primarily released from dead cells because 1) supernatantsfrom cells transfected with the p55 heavy chain fusion gene and no lightchain gene did not have detectable fusion protein until after there wassignificant cell death, and 2) the ratio of the k− to k+versions ofp55-sf2 increased as cell cultures went from 95% viability to 10%viability.

EXAMPLE XXVI p75

[0544] To make a p75 heavy chain fusion (p75-sf2), amino acids 1-235(Smith et al., Science 248: 1019-1023 (1990) and Kohno et al., Proc.Natl. Acad. Sci. 87:8331-8335 (1990)) were encoded in a fragmentprepared using the 5′ oligo 5′CACAGCTGCCCGCCCAGGTGGCAT-3′ (SEQ ID NO:17)(with the Leu¹ codon) and the 3′ oligo 5′-GTCGCCAGTGCTCCC TT-3′ (SEQ IDNO: 18) (with the complement of the Asp²³⁵ codon). Two other p75 heavychain fusions (p75P-sf2 and p75P-sf3) were made using the same 5′ oligowith the 3′ oligo 5′ATCGGACGTGGACGTGCAGA-3′ (SEQ ID NO:19). Theresulting PCR fragment encoded amino acids 1-182. The PCR fragments wereblunt-end ligated into the Stul or EcoRV site of the appropriate vectorand checked for the absence of errors by sequencing the insertscompletely.

[0545] Several different versions of the p75 fusion proteins were alsoexpressed. p75-sf2 has the complete extracellular domain of p75 fused tothe heavy chain while p75P-sf2 lacks the C-terminal 53 amino acids ofthe p75 extracellular domain. p75P-sB3 is the same as p75P-sf2 exceptthat it lacks the C_(H)I domain. The region deleted in p75P-sf2 and -sf3contains sites of O-linked glycosylation and a proline-rich region,neither of which is present in the extracellular domain of p55.Seckinger et al., Proc. Nat. Acad. Sci. USA 87:5188-5192 (1990).

[0546] Similar to p55-sf2, two bands were seen for p75-sf2 (lane 7) andp75P-sf2 (lane 8).

WEHI Cytotoxicity Assays

[0547] The ability of the various fusion proteins to bind and neutralizehuman TNFα or TNFβ was tested in a TNF-mediated cell killing assay.Overnight incubation of the murine fibrosarcoma cell line, WEHI 164(Espevik et al.,J. Immunol. Methods 95:99-105 (1986)), with 20 pM (1ng/ml) TNFα results in essentially complete death of the culture. Whenthe fusion proteins were pre-incubated with TNFα (FIG. 31A, B and C andTable 1 above) or TNFβ (FIG. 32) and the mixture added to cells, eachfusion protein demonstrated dose-dependent protection of the cells fromTNF cytotoxicity. Comparison of the viability of control cells notexposed to TNF to cells incubated in both TNF and fusion protein showedthat the protection was essentially complete at higher concentrations offusion protein.

[0548] Tetravalent p55-df2 showed the greatest affinity for TNFαrequiring a concentration of only 55 pM to confer 50% inhibition of 39pM (2 ng/ml) TNFα (FIG. 31A and Table 1). Bivalent p55-sf2 and p75P-sf2were nearly as efficient, requiring concentrations of 70 pM tohalf-inhibit TNFα. Approximately two times as much p75-sf2 was requiredto confer 50% inhibition compared to p55-sf2 at the TNF concentrationthat was used. The monomeric, non-fusion form of p55 was much lessefficient at inhibiting TNFα requiring a 900-fold molar excess over TNFαto inhibit cytotoxicity by 50%. This much-reduced inhibition was alsoobserved with a monomeric, Fab-like p55 fusion protein that was requiredat a 2000-fold molar excess over TNFα to get 50% inhibition. The orderof decreasing inhibitory activity was thereforep55-df2 >p55-sf2=p75P-sf2>p75-sf2>>>monomeric p55.

[0549] Surprisingly, the order of decreasing inhibitory activity wasdifferent for TNFβ, as presented in FIG. 32. p75P-sf2 was most efficientat inhibition requiring a concentration of 31 pM to half-inhibit humanTNFβ at 2 pM. Compared to p75P-sf2, three times as much p75-sf2 andthree times as much p55-sf2 were necessary to obtain the same degree ofinhibition. The order of decreasing inhibitory activity was thereforep75P-sf2>p75-sf2=p55-sf2.

Affinity measurements

[0550] A comparison was made of the binding affinity of various fusionproteins and TNFα by saturation binding (FIGS. 33A and 33B) andScatchard analysis (FIGS. 33C-33H). A microtiter plate was coated withexcess goat anti-Fc polyclonal antibody and incubated with 10 ng/ml offusion protein in TBST buffer (10 mM Tris-HCl, pH 7.8, 150 NaCl, 0.05%Tween-20) for 1 hour. Varying amounts of ¹²⁵I labeled TNFα (specificactivity—34.8 μCi/μg) was then incubated with the captured fusionprotein in PBS (10 mM Na Phosphate, pH 7.0, 150 mM NaCl) with 1% bovineserum albumin for 2 hours. Unbound TNFα was washed away with four washesin PBS and the cpm bound was quantitated using a y-counter. All sampleswere analyzed in triplicate. The slope of the lines in (FIGS. 33C-H)represent the affinity constant, K_(a). The dissociation constant(K_(d)) values (see Table 1) were derived using the equation K_(d)=1/K.

EXAMPLE XXVIII In Vivo Results

[0551] C3H mice were challenged with 5 μg of human TNFα after treatmentwith an immunoreceptor molecule of the invention. The effect of thetreatment was compared with two control treatments. The first control,cA2, is a chimeric mouse/human IgG₁ monoclonal antibody that binds humanTNF, and thus is a positive control. The second control, c17-1A, is achimeric mouse/human IgG₁ irrelevant monoclonal antibody and is thus anegative control. The results of the treatments were as presented in thefollowing Table 19. TABLE 19 Treatment Dead Fraction % Dead 1 μg cA25/14 36% 10 μg cA2 1/15 7% 50 μg c17-1A 13/15  87% 1 μg p55-sf2 8/15 53%10 μg p55-sf2 0/15 0% 50 μg p55-sf2 0/15 0%

[0552] Mice were injected with 25 μg of p55 fusion protein or a controlantibody and 1 hour later were challenged with 1 μg lipopolysaccharide(type J5). Mice were checked 24 hourts later. The results are presentedin the following Table 20. TABLE 20 Treatment Dead Fraction % DeadControl Antibody 11/11 100% p55-sf2  0/13  0%

[0553] All references cited herein, including journal articles orabstracts, published or corresponding U.S. or foreign patentapplications, issued U.S. or foreign patents, or any other references,are entirely incorporated by reference herein, including all data,tables, figures, and text presented in the cited references.Additionally, the entire contents of the references cited within thereferences cited herein are also entirely incorporated by reference.

[0554] Reference to known method steps, conventional methods steps,known methods or conventional methods is not in any way an admissionthat any aspect, description or embodiment of the present invention isdisclosed, taught or suggested in the relevant art.

[0555] The foregoing description of the specific embodiments will sofully reveal the general nature of the invention that others can, byapplying knowledge within the skill of the art (including the contentsof the references cited herein), readily modify and/or adapt for variousapplications such specific embodiments, without undue experimentation,without departing from the general concept of the present invention.Therefore, such adaptations and modifications are intended to be withinthe meaning and range of equivalents of the disclosed embodiments, basedon the teaching and guidance presented herein. It is to be understoodthat the phraseology or terminology herein is for the purpose ofdescription and not of limitation, such that the terminology orphraseology of the present specification is to be interpreted by theskilled artisan in light of the teachings and guidance presented herein,in combination with the knowledge of one of ordinary skill in the art.

EQUIVALENTS

[0556] Those skilled in the art will know, or be able to ascertain,using no more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein. These and allother equivalents are intended to be encompassed by the followingclaims.

1 19 1 157 PRT Homo sapiens 1 Val Arg Ser Ser Ser Arg Thr Pro Ser AspLys Pro Val Ala His Val 1 5 10 15 Val Ala Asn Pro Gln Ala Glu Gly GlnLeu Gln Trp Leu Asn Arg Arg 20 25 30 Ala Asn Ala Leu Leu Ala Asn Gly ValGlu Leu Arg Asp Asn Gln Leu 35 40 45 Val Val Pro Ser Glu Gly Leu Tyr LeuIle Tyr Ser Gln Val Leu Phe 50 55 60 Lys Gly Gln Gly Cys Pro Ser Thr HisVal Leu Leu Thr His Thr Ile 65 70 75 80 Ser Arg Ile Ala Val Ser Tyr GlnThr Lys Val Asn Leu Leu Ser Ala 85 90 95 Ile Lys Ser Pro Cys Gln Arg GluThr Pro Glu Gly Ala Glu Ala Lys 100 105 110 Pro Trp Tyr Glu Pro Ile TyrLeu Gly Gly Val Phe Gln Leu Glu Lys 115 120 125 Gly Asp Arg Leu Ser AlaGlu Ile Asn Arg Pro Asp Tyr Leu Asp Phe 130 135 140 Ala Glu Ser Gly GlnVal Tyr Phe Gly Ile Ile Ala Leu 145 150 155 2 321 DNA Mus Balb/c CDS(1)...(321) 2 gac atc ttg ctg act cag tct cca gcc atc ctg tct gtg agtcca gga 48 Asp Ile Leu Leu Thr Gln Ser Pro Ala Ile Leu Ser Val Ser ProGly 1 5 10 15 gaa aga gtc agt ttc tcc tgc agg gcc agt cag ttc gtt ggctca agc 96 Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Phe Val Gly SerSer 20 25 30 atc cac tgg tat cag caa aga aca aat ggt tct cca agg ctt ctcata 144 Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile35 40 45 aag tat gct tct gag tct atg tct ggg atc cct tcc agg ttt agt ggc192 Lys Tyr Ala Ser Glu Ser Met Ser Gly Ile Pro Ser Arg Phe Ser Gly 5055 60 agt gga tca ggg aca gat ttt act ctt agc atc aac act gtg gag tct240 Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Thr Val Glu Ser 6570 75 80 gaa gat att gca gat tat tac tgt caa caa agt cat agc tgg cca ttc288 Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Ser His Ser Trp Pro Phe 8590 95 acg ttc ggc tcg ggg aca aat ttg gaa gta aaa 321 Thr Phe Gly SerGly Thr Asn Leu Glu Val Lys 100 105 3 107 PRT Mus Balb/c 3 Asp Ile LeuLeu Thr Gln Ser Pro Ala Ile Leu Ser Val Ser Pro Gly 1 5 10 15 Glu ArgVal Ser Phe Ser Cys Arg Ala Ser Gln Phe Val Gly Ser Ser 20 25 30 Ile HisTrp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile 35 40 45 Lys TyrAla Ser Glu Ser Met Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser GlySer Gly Thr Asp Phe Thr Leu Ser Ile Asn Thr Val Glu Ser 65 70 75 80 GluAsp Ile Ala Asp Tyr Tyr Cys Gln Gln Ser His Ser Trp Pro Phe 85 90 95 ThrPhe Gly Ser Gly Thr Asn Leu Glu Val Lys 100 105 4 357 DNA Mus Balb/c CDS(1)...(357) 4 gaa gtg aag ctt gag gag tct gga gga ggc ttg gtg caa cctgga gga 48 Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro GlyGly 1 5 10 15 tcc atg aaa ctc tcc tgt gtt gcc tct gga ttc att ttc agtaac cac 96 Ser Met Lys Leu Ser Cys Val Ala Ser Gly Phe Ile Phe Ser AsnHis 20 25 30 tgg atg aac tgg gtc cgc cag tct cca gag aag ggg ctt gag tgggtt 144 Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val35 40 45 gct gaa att aga tca aaa tct att aat tct gca aca cat tat gcg gag192 Ala Glu Ile Arg Ser Lys Ser Ile Asn Ser Ala Thr His Tyr Ala Glu 5055 60 tct gtg aaa ggg agg ttc acc atc tca aga gat gat tcc aaa agt gct240 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ala 6570 75 80 gtc tac ctg caa atg acc gac tta aga act gaa gac act ggc gtt tat288 Val Tyr Leu Gln Met Thr Asp Leu Arg Thr Glu Asp Thr Gly Val Tyr 8590 95 tac tgt tcc agg aat tac tac ggt agt acc tac gac tac tgg ggc caa336 Tyr Cys Ser Arg Asn Tyr Tyr Gly Ser Thr Tyr Asp Tyr Trp Gly Gln 100105 110 ggc acc act ctc aca gtc tcc 357 Gly Thr Thr Leu Thr Val Ser 1155 119 PRT Mus Balb/c 5 Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu ValGln Pro Gly Gly 1 5 10 15 Ser Met Lys Leu Ser Cys Val Ala Ser Gly PheIle Phe Ser Asn His 20 25 30 Trp Met Asn Trp Val Arg Gln Ser Pro Glu LysGly Leu Glu Trp Val 35 40 45 Ala Glu Ile Arg Ser Lys Ser Ile Asn Ser AlaThr His Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg AspAsp Ser Lys Ser Ala 65 70 75 80 Val Tyr Leu Gln Met Thr Asp Leu Arg ThrGlu Asp Thr Gly Val Tyr 85 90 95 Tyr Cys Ser Arg Asn Tyr Tyr Gly Ser ThrTyr Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Leu Thr Val Ser 115 6 8PRT Homo sapiens 6 Gly Thr Leu Val Thr Val Ser Ser 1 5 7 7 PRT Homosapiens 7 Gly Thr Lys Leu Glu Ile Lys 1 5 8 20 DNA Artificial SequencePCR oligonucleotides 8 cctggatacc tgtgaaaaga 20 9 27 DNA ArtificialSequence PCR oligonucleotides 9 cctggtacct tagtcaccgt ctcctca 27 10 27DNA Artificial Sequence PCR oligonucleotides 10 aatagatatc tccttcaacacctgcaa 27 11 21 DNA Artificial Sequence PCR oligonucleotides 11atcgggacaa agttggaaat a 21 12 16 DNA Artificial Sequence PCRoligonucleotides 12 ggcggtctgg taccgg 16 13 19 DNA Artificial SequencePCR oligonucleotides 13 gtcaacaaca tagtcatca 19 14 23 DNA ArtificialSequence PCR oligonucleotides 14 cacaggtgtg tccccaagga aaa 23 15 18 DNAArtificial Sequence PCR oligonucleotides 15 aatctggggt aggcacaa 18 16 17DNA Artificial Sequence PCR oligonucleotides 16 agtgtgtgtc cccaagg 17 1724 DNA Artificial Sequence PCR oligonucleotides 17 cacagctgcc cgcccaggtggcat 24 18 17 DNA Artificial Sequence PCR oligonucleotides 18 gtcgccagtgctccctt 17 19 20 DNA Artificial Sequence PCR oligonucleotides 19atcggacgtg gacgtgcaga 20

What is claimed is:
 1. A method of treating a myelodysplastic syndromein a human comprising administering to the human an effectiveTNF-inhibiting amount of an anti-TNF antibody, wherein said anti-TNFantibody competitively inhibits binding of TNF to monoclonal antibodycA2.
 2. A method of treating a myelodysplastic syndrome in a humancomprising administering to the human an effective TNF-inhibiting amountof an anti-TNF chimeric antibody, wherein said anti-TNF chimericantibody binds to at least one epitope included in amino acids between87-108 or both 59-80 and 87-108 of SEQ ID NO. :1 of hTNF.
 3. A method oftreating a myelodysplastic syndrome in a human comprising administeringto the human an effective TNF-inhibiting amount of chimeric anti-TNFantibody cA2.
 4. A method for treating a myelodysplastic syndrome in ahuman comprising administering to the human at least one monoclonalantibody cA2, or a TNF binding fragment thereof.
 5. A method of treatinga myelodysplastic syndrome in a human comprising administering to thehuman an effective TNF-inhibiting amount of an anti-TNF chimericantibody, wherein said anti-TNF chimeric antibody comprises an IgG1constant region and competitively inhibits binding of TNF to monoclonalantibody cA2.
 6. A method of treating a myelodysplastic syndrome in ahuman comprising administering to the human an effective TNF-inhibitingamount of an anti-TNF chimeric antibody, wherein said anti-TNF chimericantibody comprises an IgG1 constant region and binds to at least oneepitope included in amino acids between 87-108 or both 59-80 and 87-108of SEQ ID NO.:1 of hTNF.
 7. A method of treating a myelodysplasticsyndrome in a human comprising administering to the human an effectiveTNF-inhibiting amount of an anti-TNF chimeric antibody, wherein saidanti-TNF chimeric antibody comprises a non-human variable regioncomprising an amino acid sequence selected from the group consisting ofSEQ ID NO.:3 and SEQ ID NO.:5.
 8. A method of treating a myelodysplasticsyndrome in a human comprising administering to the human an effectiveTNF-inhibiting amount of an anti-TNF chimeric antibody, wherein saidanti-TNF chimeric antibody comprises a non-human variable regioncomprising an amino acid sequence selected from the group consisting ofSEQ ID NO.:3 and SEQ ID NO.:5 and an IgG1 human constant region.
 9. Themethod of claim 7 wherein the non-human variable region comprises apolypeptide encoded by a nucleic acid sequence selected from the groupconsisting of SEQ ID NO.:2 and SEQ ID NO.:4.
 10. The method of claim 8wherein the non-human variable region comprises a polypeptide encoded bya nucleic acid sequence selected from the group consisting of SEQ IDNO.:2 and SEQ ID NO.:
 4. 11. A method of treating a myelodysplasticsyndrome in a human comprising administering to the human an effectiveTNF-inhibiting amount of an anti-TNF chimeric antibody, wherein saidanti-TNF chimeric antibody competitively inhibits binding of TNF tomonoclonal antibody cA2.
 12. The method of claim 1 wherein said anti-TNFantibody is a humanized antibody.
 13. The method of claim 1 wherein saidanti-TNF antibody is a human antibody.